Molecular targets and compounds, and methods to identify the same, useful in the treatment of bone and joint degenerative diseases

ABSTRACT

The present invention relates to methods for identifying agents capable of inhibiting the expression or activity of proteins involved in the processes modulating osteoclastogenesis, which inhibition is useful in the prevention and/or treatment of bone and joint degenerative diseases and diseases involving aberrant activity or differentiation of osteoclasts. In particular, the present invention provides methods for identifying agents for use in the prevention and/or treatment of rheumatoid arthritis.

RELATED APPLICATION

The present application claims the benefit under 35 U.S.C. §119 of U.S.Provisional application No. 936,569, filed Jun. 20, 2007, and the entiredisclosure of said application is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

The present invention relates to methods for identifying agents capableof inhibiting the expression or activity of proteins involved in theprocesses modulating osteoclastogenesis, which inhibition is useful inthe prevention and/or treatment of bone and joint degenerative diseasesand diseases involving aberrant activity of osteoclasts. In particular,the present invention provides methods for identifying agents for use inthe prevention and/or treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic joint degenerative disease,characterized by inflammation and destruction of the joint structures.When the disease is unchecked, it leads to substantial disability andpain due to the loss of joint functionality and even premature death.The aim of an RA therapy, therefore, is not to slow down the disease butto attain remission in order to stop the joint destruction. Besides theseverity of the disease outcome, the high prevalence of RA (˜0.8% ofadults are affected worldwide) means a high socio-economic impact (Forreviews on RA, we refer to Smolen and Steiner (2003); Lee and Weinblatt(2001); Choy and Panayi (2001); O'Dell (2004) and Firestein (2003)).

Histological analysis of the joints of RA patients clearly evidences themechanisms involved in the RA-associated degradative processes. Thesynovium is a cell layer, composed of a sublining and a lining regionthat separates the joint capsule from the synovial cavity. The inflamedsynovium is central to the pathophysiology of RA. The synovial joint isshown as composed of two adjacent bony ends each covered with a layer ofcartilage, separated by a joint space and surrounded by the synovialmembrane and joint capsule. The synovial membrane is composed of thesynovial lining (facing the cartilage and bone), which consists of athin (1-3 cells) layer of synoviocytes and the sublining connectivetissue layer that is highly vascularised. Histological differences inthe synovium between normal and RA patients are indicated in FIG. 1.

Like many other forms of arthritis, rheumatoid arthritis (RA) isinitially characterized by an inflammatory response of the synovialmembrane (‘synovitis’) that is characterized by an important influx ofvarious types of mononuclear cells as well as by the activation of thelocal or infiltrated mononuclear cells. The lining layer becomeshyperplastic (it can have a thickness of >20 cells) and the synovialmembrane expands. However, in addition, the hallmark of RA is jointdestruction: the joint spaces narrow or disappear as a sign of cartilagedegradation and destructions of the adjacent bone, also termed‘erosions’, have occurred. The destructive portion of the synovialmembrane is termed ‘pannus’. Various forms of bone degradation areapparent in RA. Besides a generalized osteoporosis, RA is alsocharacterized by the erosion of the bone under and adjacent to thecartilage. These focal erosions result principally from the presence ofan increased population of osteoclasts at the interface of bone andpannus (for a review on bone degradation in RA, we refer to Gravallese,2002). Osteoclasts are multinucleated cells that attach to bone andsecrete bone matrix degrading enzymes (e.g. Cathepsin K, MMP9) in anacidified space between the cell and the bone tissue (the resorptionlacuna). In healthy individuals, the remodeling of bone is controlled bythe activity of these osteoclasts, which resorb bone, and the activityof osteoblasts, which are involved in the production of the calcifiedbone matrix. Osteoblasts differentiate from mesenchymal stem cells,while osteoclasts differentiate from hematopoietic monocyte/macrophageprecursors.

In RA, the concentration of the factors inducing osteoclastdifferentiation is increased at the interface between bone and thepannus (Pettit et al., 2006), leading to the dysregulation of thebalance between bone formation and bone degradation. Key players inosteoclast differentiation are the receptor activator of NF-κB (RANK)and its ligand (RANKL) and osteoprotegerin (OPG).

RANKL is a membrane-anchored ligand of the TNF superfamily In normalbone tissue, RANKL is expressed by osteoblasts, but in RA, synovialfibroblasts as well as activated T lymphocytes are important sources ofRANKL. RANKL exerts its effect on osteoclasts or osteoclast precursorcells through RANK, a member of the TNF receptor superfamily. Anotherkey player in osteoclast biology is OPG, a RANKL decoy receptor, whichbelongs to the TNF receptor superfamily and competes with RANK for thebinding of RANKL. OPG, therefore, effectively inhibits osteoclastmaturation and osteoclast activation. OPG-transgenic mice have a highbone mass (osteopetrosic phenotype), whereas the absence of OPG resultsin severe osteoporosis, as shown in OPG-knockout mice (Bucay et al.,1998). In summary, the balance between RANK/RANKL signaling and levelsof OPG, the soluble decoy receptor for RANKL, regulates the developmentand activation of osteoclasts and therefore is strongly involved in bonemetabolism. Thus, inhibition of RANKL function via OPG might preventbone destruction in several diseases, e.g., RA. Of significance in thisrespect is the observation that RANKL knock-out mice are less prone tobone erosion when subjected to CIA (Pettit et al., 2001) and thatrecombinant OPG, alone or in combination with an anti-TNFα, preventsbone erosions in animal models for RA (Redlich et al., 2004). Inaddition, the capacity of drugs inducing OPG expression to protect bonein animal models of arthritis, in PTH induced bone resorption in ratsand in metastasis of breast cancer cells to bone has been demonstrated(Onyia et al., 2004).

From the description of the biology of RANK, RANKL and OPG, it is clearthat influencing the activity or differentiation of osteoclasts throughmodulation of these factors has potential not only in RA, but also forthe treatment of osteoporosis. In addition, as bone metastasisassociated with cancer also requires bone remodeling, inhibitors ofosteoclast activity or differentiation could also be of use for thisindication. For a review on bone metastasis, see Roodman, 2004.

Reported Developments

NSAIDS (Non-steroidal anti-inflammatory drugs) are used to reduce thepain associated with RA and improve life quality of the patients. Thesedrugs will not, however, put a brake on the RA-associated jointdestruction.

Corticosteroids are found to decrease the progression of RA as detectedradiographically and are used at low doses to treat a subset of RApatients (30 to 60%). Serious side effects, however, are associated withlong corticosteroid use (e.g. skin thinning, osteoporosis, cataracts,hypertension, hyperlipidemia).

Synthetic Disease-Modifying Anti-Rheumatic Drugs (DMARDs, e.g.methotrexate, leflunomide, sulfasalazine) mainly tackle theimmuno-inflammatory component of RA. As a main disadvantage, these drugsonly have a limited efficacy (joint destruction is only slowed down butnot blocked by DMARDs such that disease progression in the long termcontinues). The lack of efficacy is indicated by the fact that, onaverage, only 30% of the patients achieve an ACR50 score after 24 monthstreatment with methotrexate, meaning that, according to the AmericanCollege of Rheumatology, only 30% of the patients achieve a 50%improvement of their symptoms (O'Dell et al., 1996). In addition, theprecise mechanism of action of DMARDs is often unclear.

Biological DMARDs (Infliximab, Etanercept, Adalimumab, Rituximab,CTLA4-Ig) are therapeutic proteins that inactivate cytokines (forexample, TNF-α) or cells (for example, T-cells or B-cells) that have animportant role in the RA pathophysiology (Kremer et al., 2003; Edwardset al., 2004). Although the TNF-α-blockers (Infliximab, Etanercept,Adalimumab) and methotrexate combination therapy is the most effectiveRA treatment currently available, it is striking that even this therapyonly achieves a 50% improvement (ACR50) in disease symptoms in 50-60% ofpatients after 12 months therapy (St Clair et al., 2004). Increased riskof infections (tuberculosis), hematologic events and demyelinatingdisorders have been described for the TNF-α blockers (see alsoGomez-Reino et al., 2003). TNF-α blockers, which are biologicaltherapies, also require an unpleasant method of administration (frequentinjections accompanied by infusion site reactions) and have highproduction cost. The fact that a variety of targeted therapies havesimilar but limited efficacies, suggests that there is a multiplicity ofpathogenic factors for RA.

This calls for additional strategies to achieve remission. Remission isrequired since residual disease bears the risk of progressive jointdamage and thus progressive disability Inhibiting theimmuno-inflammatory component of the RA disease, which represents themain target of drugs currently used for RA treatment, does not result ina blockade of joint degradation, the major hallmark of the disease.

Additionally, bisphosphonates are inhibitors of OC activity that areaccepted as the most potent inhibitors of bone resorption clinicallyavailable and as a mainstay in the treatment of osteoporosis.Remarkably, however, bisphosphonate treatment does not preventradiologic progression in RA patients (Valleala et al., 2004),potentially because of a poor bioavailability of bisphosphonates at thelevel of the OC in the pannus. A clear need exists, therefore, forinnovative antiresorptive therapies in the field of RA.

The present invention is based on the discovery that agents whichinhibit the expression and/or activity of the TARGETS disclosed hereinare able to increase the expression of osteoprotegrin and thus haveutility in decreasing bone resorption in joints. The present inventiontherefore provides TARGETS which are involved in the pathway leading toOPG expression and osteoclastogenesis, methods for screening for agentscapable of inhibiting the expression and/or activity of TARGETS and usesof these agents in the prevention and/or treatment of joint degenerativeconditions such as rheumatoid arthritis.

SUMMARY OF THE INVENTION

The present invention relates to a method for identifying compounds thatinhibit osteoclastogenesis, comprising contacting a compound with apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 41-69 and 80 (hereinafter “TARGETS”) andfragments thereof, under conditions that allow said polypeptide to bindto said compound, and measuring a compound-polypeptide property relatedto osteoclastogenesis. In a specific embodiment the compound-polypeptideproperty measured is OPG expression levels.

Aspects of the present method include the in vitro assay of compoundsusing the polypeptide corresponding to a TARGET, or fragments thereof,such fragments being fragments of the amino acid sequences described bySEQ ID NO: 41-69 and 80, and cellular assays wherein TARGET inhibitionis followed by observing indicators of efficacy including, for example,TARGET expression levels, TARGET enzymatic activity and/or OPG levels.

The present invention also relates to

-   -   (1) expression inhibitory agents comprising a polynucleotide        selected from the group of an antisense polynucleotide, a        ribozyme, and a small interfering RNA (siRNA), wherein said        polynucleotide comprises a nucleic acid sequence complementary        to, or engineered from, a naturally occurring polynucleotide        sequence encoding a TARGET polypeptide said polynucleotide        sequence comprising a sequence selected from the group        consisting of SEQ ID NO: 1-29 and 40, and    -   (2) pharmaceutical compositions comprising said agent(s), useful        in the treatment, or prevention, of chronic joint degenerative        diseases such as rheumatoid arthritis.

Another aspect of the invention is a method of treatment, or prevention,of a condition related to bone and/or joint degeneration, in a subjectsuffering or susceptible thereto, by administering a pharmaceuticalcomposition comprising an effective TARGET-expression inhibiting amountof a expression-inhibitory agent or an effective TARGET activityinhibiting amount of a activity-inhibitory agent.

A further aspect of the present invention is a method for diagnosis of acondition related to bone and/or joint degeneration comprisingmeasurement of indicators of levels of TARGET expression in a subject.

Another aspect of this invention relates to the use of agents whichinhibit a TARGET as disclosed herein in a therapeutic method, apharmaceutical composition, and the manufacture of such composition,useful for the treatment of a disease involving bone and/or jointdegeneration. In particular, the present method relates to the use ofthe agents which inhibit a TARGET in the treatment of a diseasecharacterized by osteoclastogenesis, and in particular, a diseasecharacterized by abnormal OPG expression. The agents are useful foramelioration or treatment of bone disease, particularly wherein it isdesired to reduce or control osteoclast function and differentiation,including but not limited to osteoporosis, juvenile osteoporosis,osteogenesis imperfecta, hypercalcemia, hyperparathyroidism,osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis,Paget's disease of bone, bone loss due to rheumatoid arthritis,inflammatory arthritis, osteomyelitis, corticosteroid treatment,metastatic bone diseases, periodontal bone loss, bone loss due tocancer, age-related loss of bone mass, other forms of osteopenia, aswell as in instances where facilitation of bone repair or replacement isdesired such as bone fractures, bone defects, plastic surgery, dentaland other implantations. In a particular embodiment the disease isrheumatoid arthritis.

Other objects and advantages will become apparent from a considerationof the ensuing description taken in conjunction with the followingillustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic view of a normal joint and its changes in rheumatoidarthritis (From Smolen and Steiner, 2003).

FIG. 2: Example of the performance of the OPG ELISA.

FIG. 3: Schematic representation of the primary screening: Principle ofthe screening of the SilenceSelect® collection in the OPG assay.

FIG. 4: Layout and performance of the control plate used in thescreening of the SilenceSelect® collection in the OPG assay.

FIG. 5: Scatterplot representing the datapoints obtained in the primaryscreen of the OPG assay against the SilenceSelect® collection.

FIG. 6A Principle of the osteoclast—RASF coculture assay.

FIG. 6B Principle of the screening of Ad5-siRNA's in the osteoclast—RASFcoculture assay.

FIG. 7A: α_(v)β₃ and calcitonin receptor cELISA of an osteoclastmonoculture.

FIG. 7B: α_(v)β₃ and calcitonin receptor staining of a RASF-osteoclastcoculture.

FIG. 7C: Inhibition of osteoclast differentiation in a OC-RASF cocultureby adenovirus induced overexpression of OPG.

FIG. 8: Target analysis: data obtained for 6 targets

FIG. 9A: Tabulated raw data from secondary assay and MOI rescreen(screen A) for particular targets.

FIG. 9B: Tabulated raw data from secondary assay and MOI rescreen(screen B) for particular targets.

FIG. 10: Layout of the “hit plates” used for the MMP1 assay. Forselected OPG hits, the original OPG hit KD viruses as well asindependent KD viruses targeting the same genes through the expressionof different shRNAs are collected and grouped in wells C1 to F11. 3different negative control viruses (Ad5-Luc-KD_v13, Ad5-eGFP-KD_v5,Ad5-M6 PR-KD_v1) and one positive control virus (Ad5-MMP1-KD) aregrouped in rows B and G. The content of the “hit plates” is repropagatedto generate a sufficient amount of virus crude lysate for the tests andto ensure homogeneity of the titers of the viruses. During the MMP1experiments, the wells B2, B3 and B4 (shown in italics) of the platescontaining the RASFs tranduced with the content of the “hit plates” areleft untriggered, whereas all other wells are activated with “TNFalphabased trigger”

FIG. 11: Representative example of the outcome of a MMP1 assayexperiment. The normalized reduction in MMP1 expression is shown for 14KD viruses tested at 3 multiplicity of infections (MOIs) and compared tothe performance of the positive control (Ad5-MMP1-KD) and of theuntriggered or triggered negative controls. The negative control datarepresent the average of the data obtained for the 3 (untriggeredcondition) or 13 (triggered condition) negative controls present on the“hit plates”. The cutoff for hit calling is represented with a dottedline. All 14 KD viruses tested significantly reduced thecytokine-induced MMP1 expression in RASFs.

FIG. 12: Neutralization of OPG by a selected anti-OPG antibody.Pre-osteoclasts are seeded on top of RASFs in presence of indicatedamounts of recombinant OPG and anti-OPG antibody and after overnightincubation osteoclast differentiation is triggered by addition ofindicated amounts sRANKL. The number of osteoclasts formed after another11 days incubation is quantified using a vitronectin cELISA readout.Without the addition of the anti-OPG (Cat. N^(o) A805; R&D Systems),osteoclast formation is dependent on the dose of sRANKL and is blockedby the addition of OPG; the ability of OPG to prevent osteoclastformation is dependent upon the dose of sRANKL used to triggerosteoclast differentiation: the higher the dose of sRANKL, the more OPGthat is needed to prevent osteoclast differentiation. Addition of theanti-OPG antibody is able to rescue osteoclast differentiation in thepresence of OPG. The ability to rescue osteoclast differentiation isdependent upon the dose of the antibody, upon the concentration of OPGadded and upon the dose of sRANKL: the more antibody, the more OPG canbe neutralized and the lower the dose of RANKL at which rescue of OPGinhibition can be observed. As can be seen, dose-response of sRANKL whenno OPG is added is already shifted to lower sRANKL concentrations whenanti-OPG antibody is added due to neutralization of endogenous secretedOPG by RASFs. For the experiments, the anti-OPG Ab is used at aconcentration of 3 μg/mL and 15 ng/mL sRANKL is used to triggerosteoclast differentiation.

DETAILED DESCRIPTION

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

The term ‘agent’ means any molecule, including polypeptides, antibodies,polynucleotides, chemical compounds and small molecules. In particularthe term agent includes compounds such as test compounds or drugcandidate compounds.

The term ‘agonist’ refers to a ligand that stimulates the receptor theligand binds to in the broadest sense.

The term ‘assay’ means any process used to measure a specific propertyof a compound. A ‘screening assay’ means a process used to characterizeor select compounds based upon their activity from a collection ofcompounds.

The term ‘binding affinity’ is a property that describes how stronglytwo or more compounds associate with each other in a non-covalentrelationship. Binding affinities can be characterized qualitatively,(such as ‘strong’, ‘weak’, ‘high’, or ‘low’) or quantitatively (such asmeasuring the KO.

The term ‘carrier’ means a non-toxic material used in the formulation ofpharmaceutical compositions to provide a medium, bulk and/or useableform to a pharmaceutical composition. A carrier may comprise one or moreof such materials such as an excipient, stabilizer, or an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude aqueous or solid buffer ingredients including phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

The term ‘complex’ means the entity created when two or more compoundsbind to, contact, or associate with each other.

The term ‘compound’ is used herein in the context of a ‘test compound’or a ‘drug candidate compound’ described in connection with the assaysof the present invention. As such, these compounds comprise organic orinorganic compounds, derived synthetically or from natural sources. Thecompounds include inorganic or organic compounds such aspolynucleotides, lipids or hormone analogs. Other biopolymeric organictest compounds include peptides comprising from about 2 to about 40amino acids and larger polypeptides comprising from about 40 to about500 amino acids, including polypeptide ligands, enzymes, receptors,channels, antibodies or antibody conjugates.

The term ‘condition’ or ‘disease’ means the overt presentation ofsymptoms (i.e., illness) or the manifestation of abnormal clinicalindicators (for example, biochemical indicators or diagnosticindicators). Alternatively, the term ‘disease’ refers to a genetic orenvironmental risk of or propensity for developing such symptoms orabnormal clinical indicators.

The term ‘contact’ or ‘contacting’ means bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

The term ‘derivatives of a polypeptide’ relates to those peptides,oligopeptides, polypeptides, proteins and enzymes that comprise astretch of contiguous amino acid residues of the polypeptide and thatretain a biological activity of the protein, for example, polypeptidesthat have amino acid mutations compared to the amino acid sequence of anaturally-occurring form of the polypeptide. A derivative may furthercomprise additional naturally occurring, altered, glycosylated, acylatedor non-naturally occurring amino acid residues compared to the aminoacid sequence of a naturally occurring form of the polypeptide. It mayalso contain one or more non-amino acid substituents, or heterologousamino acid substituents, compared to the amino acid sequence of anaturally occurring form of the polypeptide, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence.

The term ‘derivatives of a polynucleotide’ relates to DNA-molecules,RNA-molecules, and oligonucleotides that comprise a stretch of nucleicacid residues of the polynucleotide, for example, polynucleotides thatmay have nucleic acid mutations as compared to the nucleic acid sequenceof a naturally occurring form of the polynucleotide. A derivative mayfurther comprise nucleic acids with modified backbones such as PNA,polysiloxane, and 2′-O-(2-methoxy) ethyl-phosphorothioate, non-naturallyoccurring nucleic acid residues, or one or more nucleic acidsubstituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,amino-, propyl-, chloro-, and methanocarbanucleosides, or a reportermolecule to facilitate its detection.

The term ‘osteoclast’ refers to the large multinucleate cells found ingrowing bone that resorbs bony tissue, as in the formation of canals andcavities.

The term costeoclastogenesis' refers to the process by which osteoclastsare generated by fusion of cells of the monocyte-macrophage cell line.

The term ‘effective amount’ or ‘therapeutically effective amount’ meansthat amount of a compound or agent that will elicit the biological ormedical response of a subject that is being sought by a medical doctoror other clinician.

The term ‘endogenous’ shall mean a material that a mammal naturallyproduces. Endogenous in reference to the term ‘protease’, ‘kinase’, orG-Protein Coupled Receptor (‘GPCR’) shall mean that which is naturallyproduced by a mammal (for example, and not limitation, a human) Incontrast, the term non-endogenous in this context shall mean that whichis not naturally produced by a mammal (for example, and not limitation,a human). Both terms can be utilized to describe both in vivo and invitro systems. For example, and without limitation, in a screeningapproach, the endogenous or non-endogenous TARGET may be in reference toan in vitro screening system. As a further example and not limitation,where the genome of a mammal has been manipulated to include anon-endogenous TARGET, screening of a candidate compound by means of anin vivo system is viable.

The term ‘expressible nucleic acid’ means a nucleic acid coding for aproteinaceous molecule, an RNA molecule, or a DNA molecule.

The term ‘expression’ comprises both endogenous expression andoverexpression by transduction.

The term ‘expression inhibitory agent’ means a polynucleotide designedto interfere selectively with the transcription, translation and/orexpression of a specific polypeptide or protein normally expressedwithin a cell. More particularly, ‘expression inhibitory agent’comprises a DNA or RNA molecule that contains a nucleotide sequenceidentical to or complementary to at least about 15-30, particularly atleast 17, sequential nucleotides within the polyribonucleotide sequencecoding for a specific polypeptide or protein. Exemplary expressioninhibitory molecules include ribozymes, double stranded siRNA molecules,self-complementary single-stranded siRNA molecules, genetic antisenseconstructs, and synthetic RNA antisense molecules with modifiedstabilized backbones.

The term ‘fragment of a polynucleotide’ relates to oligonucleotides thatcomprise a stretch of contiguous nucleic acid residues that exhibitsubstantially a similar, but not necessarily identical, activity as thecomplete sequence. In a particular aspect, ‘fragment’ may refer to aoligonucleotide comprising a nucleic acid sequence of at least 5 nucleicacid residues (preferably, at least 10 nucleic acid residues, at least15 nucleic acid residues, at least 20 nucleic acid residues, at least 25nucleic acid residues, at least 40 nucleic acid residues, at least 50nucleic acid residues, at least 60 nucleic residues, at least 70 nucleicacid residues, at least 80 nucleic acid residues, at least 90 nucleicacid residues, at least 100 nucleic acid residues, at least 125 nucleicacid residues, at least 150 nucleic acid residues, at least 175 nucleicacid residues, at least 200 nucleic acid residues, or at least 250nucleic acid residues) of the nucleic acid sequence of said completesequence.

The term ‘fragment of a polypeptide’ relates to peptides, oligopeptides,polypeptides, proteins, monomers, subunits and enzymes that comprise astretch of contiguous amino acid residues, and exhibit substantially asimilar, but not necessarily identical, functional or expressionactivity as the complete sequence. In a particular aspect, ‘fragment’may refer to a peptide or polypeptide comprising an amino acid sequenceof at least 5 amino acid residues (preferably, at least 10 amino acidresidues, at least 15 amino acid residues, at least 20 amino acidresidues, at least 25 amino acid residues, at least 40 amino acidresidues, at least 50 amino acid residues, at least 60 amino residues,at least 70 amino acid residues, at least 80 amino acid residues, atleast 90 amino acid residues, at least 100 amino acid residues, at least125 amino acid residues, at least 150 amino acid residues, at least 175amino acid residues, at least 200 amino acid residues, or at least 250amino acid residues) of the amino acid sequence of said completesequence.

The term ‘hybridization’ means any process by which a strand of nucleicacid binds with a complementary strand through base pairing. The term‘hybridization complex’ refers to a complex formed between two nucleicacid sequences by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(for example, C_(0t) or R_(0t) analysis) or formed between one nucleicacid sequence present in solution and another nucleic acid sequenceimmobilized on a solid support (for example, paper, membranes, filters,chips, pins or glass slides, or any other appropriate substrate to whichcells or their nucleic acids have been fixed). The term “stringentconditions” refers to conditions that permit hybridization betweenpolynucleotides and the claimed polynucleotides. Stringent conditionscan be defined by salt concentration, the concentration of organicsolvent, for example, formamide, temperature, and other conditions wellknown in the art. In particular, reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature can increase stringency. The term ‘standard hybridizationconditions’ refers to salt and temperature conditions substantiallyequivalent to 5×SSC and 65° C. for both hybridization and wash. However,one skilled in the art will appreciate that such ‘standard hybridizationconditions’ are dependent on particular conditions including theconcentration of sodium and magnesium in the buffer, nucleotide sequencelength and concentration, percent mismatch, percent formamide, and thelike. Also important in the determination of “standard hybridizationconditions” is whether the two sequences hybridizing are RNA-RNA,DNA-DNA or RNA-DNA. Such standard hybridization conditions are easilydetermined by one skilled in the art according to well known formulae,wherein hybridization is typically 10-20^(N) C below the predicted ordetermined T_(m) with washes of higher stringency, if desired.

The term ‘inhibit’ or ‘inhibiting’, in relationship to the term‘response’ means that a response is decreased or prevented in thepresence of a compound as opposed to in the absence of the compound.

The term ‘inhibition’ refers to the reduction, down regulation of aprocess or the elimination of a stimulus for a process, which results inthe absence or minimization of the expression or activity of a proteinor polypeptide.

The term ‘induction’ refers to the inducing, up-regulation, orstimulation of a process, which results in the expression or activity ofa protein or polypeptide.

The term ‘ligand’ means an endogenous, naturally occurring moleculespecific for an endogenous, naturally occurring receptor.

The term ‘pharmaceutically acceptable salts’ refers to the non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds which inhibit the expression or activity of TARGETS asdisclosed herein. These salts can be prepared in situ during the finalisolation and purification of compounds useful in the present invention.

The term ‘polypeptide’ relates to proteins (such as TARGETS),proteinaceous molecules, fragments of proteins, monomers, subunits orportions of polymeric proteins, peptides, oligopeptides and enzymes(such as kinases, proteases, GPCR's etc.).

The term ‘polynucleotide’ means a polynucleic acid, in single or doublestranded form, and in the sense or antisense orientation, complementarypolynucleic acids that hybridize to a particular polynucleic acid understringent conditions, and polynucleotides that are homologous in atleast about 60 percent of its base pairs, and more particularly 70percent of its base pairs are in common, most particularly 90 percent,and in a particular embodiment, 100 percent of its base pairs. Thepolynucleotides include polyribonucleic acids, polydeoxyribonucleicacids, and synthetic analogues thereof. It also includes nucleic acidswith modified backbones such as peptide nucleic acid (PNA),polysiloxane, and 2′-O-(2-methoxy)ethylphosphorothioate. Thepolynucleotides are described by sequences that vary in length, thatrange from about 10 to about 5000 bases, particularly about 100 to about4000 bases, more particularly about 250 to about 2500 bases. Onepolynucleotide embodiment comprises from about 10 to about 30 bases inlength. A particular embodiment of polynucleotide is thepolyribonucleotide of from about 17 to about 22 nucleotides, morecommonly described as small interfering RNAs (siRNAs). Anotherparticular embodiment are nucleic acids with modified backbones such aspeptide nucleic acid (PNA), polysiloxane, and2′-O-(2-methoxy)ethylphosphorothioate, or including non-naturallyoccurring nucleic acid residues, or one or more nucleic acidsubstituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,amino-, propyl-, chloro-, and methanocarbanucleosides, or a reportermolecule to facilitate its detection. Polynucleotides herein areselected to be ‘substantially’ complementary to different strands of aparticular target DNA sequence. This means that the polynucleotides mustbe sufficiently complementary to hybridize with their respectivestrands. Therefore, the polynucleotide sequence need not reflect theexact sequence of the target sequence. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the polynucleotide,with the remainder of the polynucleotide sequence being complementary tothe strand. Alternatively, non-complementary bases or longer sequencescan be interspersed into the polynucleotide, provided that thepolynucleotide sequence has sufficient complementarity with the sequenceof the strand to hybridize therewith under stringent conditions or toform the template for the synthesis of an extension product.

The term ‘preventing’ or ‘prevention’ refers to a reduction in risk ofacquiring or developing a disease or disorder (i.e., causing at leastone of the clinical symptoms of the disease not to develop) in a subjectthat may be exposed to a disease-causing agent, or predisposed to thedisease in advance of disease onset.

The term ‘prophylaxis’ is related to ‘prevention’, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

The term ‘solvate’ means a physical association of a compound useful inthis invention with one or more solvent molecules. This physicalassociation includes hydrogen bonding. In certain instances the solvatewill be capable of isolation, for example when one or more solventmolecules are incorporated in the crystal lattice of the crystallinesolid. “Solvate” encompasses both solution-phase and isolable solvates.Representative solvates include hydrates, ethanolates and methanolates.

The term ‘subject’ includes humans and other mammals.

The term ‘TARGET’ or ‘TARGETS’ means the protein(s) or polypeptide(s)identified in accordance with the assays described herein and determinedto be involved in the modulation of OPG expression levels.

‘Therapeutically effective amount’ means that amount of a drug,compound, expression inhibitory agent, or pharmaceutical agent that willelicit the biological or medical response of a subject that is beingsought by a medical doctor or other clinician. In particular, withregard to treating an disease condition characterized by the activationof osteoclastogenesis, the term ‘effective bone resorption inhibitingamount’ is intended to mean that effective amount of a compound thatinhibits a TARGET as disclosed herein that will bring about abiologically meaningful increase in the expression of OPG in thesubject's disease affected tissues such that osteoclastogenesis isinhibited and bone resorption is reduced.

A compound having OPG inducing properties or an ‘OPG inducing compound’means a compound that when provided to a cell in effective amounts isable to cause a biologically meaningful increase in the expression orproduction of OPG in such cells.

The term ‘treating’ or ‘treatment’ of any disease or disorder refers, inone embodiment, to ameliorating the disease or disorder (i.e., arrestingthe disease or reducing the manifestation, extent or severity of atleast one of the clinical symptoms thereof). In another embodiment‘treating’ or ‘treatment’ refers to ameliorating at least one physicalparameter, which may not be discernible by the subject. In yet anotherembodiment, ‘treating’ or ‘treatment’ refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In a further embodiment, ‘treating’ or ‘treatment’relates to slowing the progression of the disease.

Applicants' invention is relevant to the reduction of bone resorptionand osteoclastogenesis, and is in part based on the TARGET'srelationship to OPG expression and osteoclast differentiation. TheTARGETs are relevant in bone and joint diseases which involve or invokebone resorption or degradation. In particular, the TARGETs are relevantin rheumatoid arthritis.

OPG expression is relevant to bone resorption as it inversely correlatesto the stimulation of osteoclastogenesis towards an activated phenotypethat, in vivo, is responsible for bone resorption. This is supported bythe observation that RANKL knock-out mice are less prone to bone erosionwhen subjected to CIA (Pettit et al., 2001) and recombinant OPG, aloneor in combination with an anti-TNFα, prevents bone erosions in animalmodels for RA (Redlich et al., 2004).

Therefore, induction of OPG expression represents a valuable therapeuticapproach towards the treatment of RA. Accordingly, if the reduction inexpression of a candidate protein in synovial fibroblasts or anothercell composing the joint leads to an increased in OPG expression and/oractivity levels, then such protein is involved in the regulation of OPGexpression and is a relevant target for the development of therapeuticstrategies for the treatment of RA. The present inventors haveidentified such target proteins by screening recombinant adenovirusesmediating the expression of a library of shRNAs, referred to herein as‘Ad-siRNAs’. The collection used herein is further referred to as an‘adenoviral siRNA library’ or the SilenceSelect® collection. Theselibraries contain recombinant adenoviruses, further referred to asknock-down (KD) viruses or Ad-siRNAs, that mediate the expression incells of shRNAs which reduce the expression levels of targeted genes bya RNA interference (RNAi)-based mechanism (WO03/020931). The screeningwork is described below in Example 1.

As noted above, the present invention is based on the present inventors'discovery that the TARGET polypeptides, identified as a result of avariety of screens described below in the Examples, are factors not onlyin the regulation of expression of OPG, but also in the modulation ofosteoclastogenesis. A reduced activity of the OPG-inducing proteins isbelieved to be causative and to correlate with the progression ofvarious diseases associated with an increased bone resorption, includingdiseases that involve the degradation of the joint, e.g. rheumatoidarthritis. OPG expression is involved in bone disease, including but notlimited to osteoporosis, juvenile osteoporosis, osteogenesis imperfecta,hypercalcemia, hyperparathyroidism, osteomalacia, osteohalisteresis,osteolytic bone disease, osteonecrosis, Paget's disease of bone, boneloss due to rheumatoid arthritis, inflammatory arthritis, osteomyelitis,corticosteroid treatment, metastatic bone diseases, periodontal boneloss, bone loss due to cancer, age-related loss of bone mass, otherforms of osteopenia. Modulation of OPG may also be useful in instanceswhere facilitation of bone repair or replacement is desired such as bonefractures, bone defects, plastic surgery, dental and otherimplantations.

In one aspect, the present invention relates to a method for assayingfor drug candidate compounds that inhibit bone resorption, comprisingcontacting the compound with a polypeptide comprising an amino acidsequence of SEQ ID NO: 41-69 and 80, or fragment thereof, underconditions that allow said polypeptide to bind to the compound, anddetecting the formation of a complex between the polypeptide and thecompound. One particular means of measuring the complex formation is todetermine the binding affinity of said compound to said polypeptide.

More particularly, the invention relates to a method for identifying anagent that inhibits bone resorption, the method comprising:

-   -   (a) contacting a population of mammalian cells with one or more        compound that exhibits binding affinity for a TARGET        polypeptide, or fragment thereof, and    -   (b) measuring a compound-polypeptide property related to bone        resorption.

In a further aspect, the present invention relates to a method forassaying for drug candidate compounds that inhibit bone resorption,comprising contacting the compound with a polypeptide comprising anamino acid sequence of SEQ ID NO: 41-69 and 80, or fragment thereof,under conditions that allow said compound to modulate the activity orexpression of the polypeptide, and determining the activity orexpression of the polypeptide. One particular means of measuring theactivity or expression of the polypeptide is to determine the amount ofsaid polypeptide using a polypeptide binding agent, such as an antibody,or to determine the activity of said polypeptide in a biological orbiochemical measure, for instance the amount of phosphorylation of atarget of a kinase polypeptide.

The compound-polypeptide property referred to above is related to theexpression and/or activity of the TARGET, and is a measurable phenomenonchosen by the person of ordinary skill in the art. The measurableproperty may be, for example, the binding affinity of said compound fora peptide domain of the polypeptide TARGET or the level of any one of anumber of biochemical marker levels of bone resorption. An event oractivity related to bone resorption can be measured, for example, themeasurement of the amount or activity of osteoclasts or the measurementof markers indicative for bone resorption, as e.g. CTX-I or osteocalcin.The compound may be incubated with osteoclasts, osteoclast precursors,or related cell lines and the differentiation, maturation activation andfunctional status of said cells determined There are multiple osteoclastculture systems or methods and bone formation assays that can be usedsuccessfully to screen potential osteogenic compounds of this invention.See, e.g., U.S. Pat. No. 6,080,779. One osteoclast culture for use inscreening is a neonatal mouse calvaria assay. In addition to this assay,the effect of compounds on murine calvarial bone growth can also betested in vivo. In addition, osteoclast cultures, containingmacrophages, osteoclast precursors and osteoclasts, can be generatedfrom bone marrow precursors, particularly from bone marrow macrophagesand utilized in assessment of compounds for osteoclast modulatingactivity. Bone marrow macrophages are cultured in 48- or 96-well cellculture dishes in the presence of M-CSF (10 ng/mL), RANKL (100 ng/mL),with or without addition of compound(s) or control(s), and mediumchanged (e.g. on day 3). Osteoclast-like cells are characterized bystaining for tartrate-resistant acid phosphatase (TRAP) activity. Inassessing bone resorption, for instance using a pit assay, osteoclastsare generated on whale dentin slices from bone marrow macrophages. Afterthree days of culture to generate osteoclasts, compound(s) or control(s)are added to the culture for two days. At the end of the experiment,cells are TRAP stained and photographed to document cell number. Cellsare then removed from the dentin slices with 0.5M ammonium hydroxide andmechanical agitation. Maximum resorption lacunae depth is measured usinga confocal microscope (Microradiance, Bio-Rad Laboratories, Hercules,Calif.). For evaluation of pit number and resorbed area, dentin slicesare stained with Coumassie brilliant blue and analyzed with lightmicroscopy using Osteomeasure software (Osteometrics, Decatur, Ga.) forquantitation.

In an additional aspect, the present invention relates to a method forassaying for drug candidate compounds that inhibit bone resorption,comprising contacting the compound with a nucleic acid encoding a TARGETpolypeptide, including comprising a nucleic acid sequence of SEQ ID NO:1-29 and 40, or fragment/portion thereof, under conditions that allowsaid nucleic acid to bind to or otherwise associate with the compound,and detecting the formation of a complex between the nucleic acid andthe compound. One particular means of measuring the complex formation isto determine the binding affinity of said compound to said nucleic acidor the presence of a complex by virtue of resistance to nucleases or bygel mobility assays. Alternatively, complex formation may be determinedby inhibition of nucleic acid transcription or translation.

In a particular embodiment of the invention, the TARGET polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID No: 41-69 and 80 as listed in Table 1. In an embodiment of theinvention, the nucleic acid capable of encoding the TARGET polypeptidecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1-29 and 40 as listed in Table 1.

TABLE 1 SEQ Target GenBank ID SEQ ID Gene Nucleic Acid NO: GenBank NO:Symbol Acc #: DNA Protein Acc # Protein NAME Class NTRK2 NM_006180 1NP_006171 41 neurotrophic Kinase tyrosine kinase, receptor, type 2,transcript variant a NM_001007097 2 NP_001007098 42 neurotrophictyrosine kinase, receptor, type 2, transcript variant b NM_001018064 3NP_001018074 43 neurotrophic tyrosine kinase, receptor, type 2,transcript variant c NM_001018065 4 NP_001018075 44 neurotrophictyrosine kinase, receptor, type 2, transcript variant d NM_001018066 5NP_001018076 45 neurotrophic tyrosine kinase, receptor, type 2,transcript variant e MAP4K4 NM_004834 6 NP_004825 46 mitogen-activatedKinase protein kinase kinase kinase kinase 4, transcript variant 1NM_145686 7 NP_663719 47 mitogen-activated protein kinase kinase kinasekinase 4, transcript variant 2 NM_145687 8 NP_663720 48mitogen-activated protein kinase kinase kinase kinase 4, transcriptvariant 3 SK437 9 n/a 49 kinase SK437 from Manning et al., Science.MMP17 NM_016155 10 NP_057239 50 matrix Protease metalloproteinase 17(membrane-inserted) PLA2G12A NM_030821 11 NP_110448 51 phospholipase A2,Enzyme group XIIA MGLL NM_007283 12 NP_009214 52 monoglyceride Enzymelipase, transcript variant 1 NM_001003794 13 NP_001003794 53monoglyceride lipase, transcript variant 2 GPR44 NM_004778 14 NP_00476954 G protein-coupled GPCR receptor 44 MIR16 NM_016641 15 NP_057725 55membrane PDE interacting protein of RGS16 PTK6 NM_005975 16 NP_005966 56PTK6 protein Kinase tyrosine kinase 6 MRAS NM_012219 17 NP_036351 57muscle RAS Enzyme oncogene homolog SLC4A8 NM_004858 18 NP_004849 58solute carrier family Ion 4, sodium Channel bicarbonate cotransporter,member 8 ENPP2 NM_006209 19 NP_006200 59 ectonucleotide PDEpyrophosphatase/phosphodiesterase 2 (autotaxin) MAP3K3 NM_002401 20NP_002392 60 mitogen-activated Kinase protein kinase kinase kinase 3,transcript variant 2 NM_203351 21 NP_976226 61 mitogen-activated Kinaseprotein kinase kinase kinase 3, transcript variant 1 P2RY14 NM_014879 22NP_055694 62 purinergic receptor GPCR P2Y, G-protein coupled, 14 NEK3NM_002498 23 NP_002489 63 NIMA (never in Kinase mitosis gene a)- relatedkinase 3, transcript variant 1 NM_152720 24 NP_689933 64 NIMA (never inmitosis gene a)- related kinase 3, transcript variant 2 KLKB1 NM_00089225 NP_000883 65 kallikrein B, plasma Protease (Fletcher factor) 1 FNTANM_002027 26 NP_002018 66 farnesyltransferase, Enzyme CAAX box, alpha,transcript variant 1 NM_001018676 27 NP_001018196 67farnesyltransferase, CAAX box, alpha, transcript variant 2 NM_00101867728 NP_001018197 68 farnesyltransferase, CAAX box, alpha, transcriptvariant 3 LOC283226 XM_208554 29 XP_208554 69 similar to Proteinfarnesyltransferase/geranylgeranyltransferase type I alpha subunit (CAAXfarnesyltransferase alpha subunit) (Ras proteins prenyltransferasealpha) (FTase-alpha) (Type I protein geranyl- geranyltransferase alphasubunit) (GGTase-I- . . . USP9Y NM_004654 30 NP_004645 70 ubiquitinspecific Protease peptidase 9, Y-linked (fat facets-like, Drosophila)CDC7 NM_003503 31 NP_003494 71 CDC7 cell division Kinase cycle 7 (S.cerevisiae) PPIA NM_021130 32 NP_066953 72 peptidylprolyl Enzymeisomerase A (cyclophilin A), transcript variant 1 TOP2B NM_001068 33NP_001059 73 topoisomerase Kinase (DNA) II beta 180 kDa PPP2CB NM_00415634 NP_004147 74 protein phosphatase Phosphatase 2 (formerly 2A),catalytic subunit, beta isoform, transcript variant 1 NM_001009552 35NP_001009552 75 protein phosphatase 2 (formerly 2A), catalytic subunit,beta isoform, transcript variant 2 COX10 NM_001303 36 NP_001294 76 COX10homolog, Enzyme cytochrome c oxidase assembly protein, heme A:farnesyltransferase (yeast), nuclear gene encoding mitochondrial proteinCCR1 NM_001295 37 NP_001286 77 chemokine (C-C GPCR motif) receptor 1B3GALT1 NM_020981 38 NP_066191 78 UDP- Enzyme Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 1 SLC9A8 NM_015266 39 NP_056081 79solute carrier family Ion 9 (sodium/hydrogen Channel exchanger), isoform8 CXCR6 NM_006564 40 NP_006555 80 chemokine (C—X—C GPCR motif) receptor6

Another particular embodiment of the invention comprises the TARGETSidentified as SEQ ID NOS. 56, 57, 59-61 and 80. Another particularembodiment of the invention comprises the ion channel TARGET identifiedas SEQ ID NO: 58. A further particular embodiment of the inventioncomprises the GPCR TARGETs identified as SEQ ID NOs: 54, 62 and 80. Afurther particular embodiment of the invention comprises the kinaseTARGETs identified as SEQ ID NOs: 41-49, 56, 60-61 and 63-64. A furtherparticular embodiment of the invention comprises the protease TARGETsidentified as SEQ ID NOs: 50 and 65. A further particular embodiment ofthe invention comprises the enzyme TARGETs identified as SEQ ID NOs:51-53, 57, 66-69. A further particular embodiment of the inventioncomprises the phosphodiesterase TARGETs identified as SEQ ID NOs: 55 and59. It will be appreciated by a person of skill in the art that oneprotein may have a number of reported sequences and these sequences mayinterchangeably be used to explore the same TARGET. In particular, inone embodiment the TARGET is NTRK2 which may be described by any one ofSEQ ID NOs: 41, 42, 43, 44 or 45. In a further embodiment the TARGET isMAP4K4 which may be described by any one of SEQ ID NOs: 46, 47, 48 or49. In a further embodiment the TARGET is MGLL which may be described bySEQ ID NO: 52 or 53. In a further embodiment the TARGET is MAP3K3 whichmay be described by SEQ ID NO: 60 or 61. In a further embodiment theTARGET is NEK3 which may be described by SEQ ID NO: 63 or 64. In afurther embodiment the TARGET is FNTA which may be described by any oneof SEQ ID NOs: 66, 65, 68 or 69.

Depending on the choice of the skilled artisan, the present assay methodmay be designed to function as a series of measurements, each of whichis designed to determine whether the drug candidate compound is indeedacting on the polypeptide to thereby inhibit bone resorption. Forexample, an assay designed to determine the binding affinity of acompound to the polypeptide, or fragment thereof, may be necessary, butnot sufficient, to ascertain whether the test compound would be usefulfor inhibiting bone resorption when administered to a subject.

Such binding information would be useful in identifying a set of testcompounds for use in an assay that would measure a different property,such as one further down the biochemical pathway, such as for exampleOPG expression. Such second assay may be designed to confirm that thetest compound, having binding affinity for the polypeptide, actuallyinhibits bone resorption. Such assay may be designed to confirm that thetest compound inhibits osteoclast differentiation, affects RANK orRANKL, has anti-inflammatory effects, such as effects on MMP1. Suitableand exemplary assays are known in the art and/or described furtherherein. Suitable controls should always be in place to insure againstfalse positive or false negative readings. In a particular embodiment ofthe present invention the screening method comprises the additional stepof comparing the compound to a suitable control. In one embodiment, thecontrol may be a cell or a sample that has not been in contact with thetest compound. In an alternative embodiment, the control may be a cellthat does not express the TARGET; for example in one aspect of such anembodiment the test cell may naturally express the TARGET and thecontrol cell may have been contacted with an agent, e.g. an siRNA, whichinhibits or prevents expression of the TARGET. Alternatively, in anotheraspect of such an embodiment, the cell in its native state does notexpress the TARGET and the test cell has been engineered so as toexpress the TARGET, so that in this embodiment, the control could be theuntransformed native cell.

Whilst exemplary controls are described herein, this should not be takenas limiting; it is within the scope of a person of skill in the art toselect appropriate controls for the experimental conditions being used.

The order of taking these measurements or of performing said steps isnot believed to be critical to the practice of the present invention,which may be practiced in any order. For example, one may first performa screening assay of a set of compounds for which no information isknown respecting the compounds' binding affinity for the polypeptide.Alternatively, one may screen a set of compounds identified as havingbinding affinity for a polypeptide domain, or a class of compoundsidentified as being an inhibitor of the polypeptide. However, for thepresent assay to be meaningful to the ultimate use of the drug candidatecompounds, a measurement of bone resorption activity may be necessary.Validation studies including controls and measurements of bindingaffinity to the polypeptides of the invention are nonetheless useful inidentifying a compound useful in any therapeutic or diagnosticapplication.

The present assay method may be practiced in vitro, using one or more ofthe TARGET proteins, or fragments thereof, including monomers, portionsor subunits of polymeric proteins, peptides, oligopeptides andenzymatically active portions thereof.

The binding affinity of a compound with the polypeptide TARGET can bemeasured by methods known in the art, such as using surface plasmonresonance biosensors (Biacore®), by saturation binding analysis with alabeled compound (for example, Scatchard and Lindmo analysis), bydifferential UV spectrophotometer, fluorescence polarization assay,Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescenceresonance energy transfer, and Bioluminescence resonance energytransfer. The binding affinity of compounds can also be expressed indissociation constant (Kd) or as IC₅₀ or EC₅₀. The IC₅₀ represents theconcentration of a compound that is required for 50% inhibition ofbinding of another ligand to the polypeptide. The EC₅₀ represents theconcentration required for obtaining 50% of the maximum effect in anyassay that measures TARGET function. The dissociation constant, Kd, is ameasure of how well a ligand binds to the polypeptide, it is equivalentto the ligand concentration required to saturate exactly half of thebinding-sites on the polypeptide. Compounds with a high affinity bindinghave low Kd, IC₅₀ and EC₅₀ values, for example, in the range of 100 nMto 1 pM; a moderate- to low-affinity binding relates to high Kd, IC₅₀and EC₅₀ values, for example in the micromolar range.

The present assay method may also be practiced in a cellular assay. Ahost cell expressing the TARGET, or fragment(s) thereof, can be a cellwith endogenous expression or a cell over-expressing the TARGET, forexample, by transduction. When the endogenous expression of thepolypeptide is not sufficient to determine a baseline that can easily bemeasured, one may use host cells that over-express TARGET.Over-expression has the advantage that the level of the TARGET substrateend-products is higher than the activity level by endogenous expression.Accordingly, measuring such levels using presently available techniquesis easier. Alternatively, a non-endogenous form of TARGET may beexpressed or overexpressed in a cell and utilized in screening.

One embodiment of the present method for identifying a compound thatincreases OPG expression and/or activity comprises culturing apopulation of mammalian cells expressing a TARGET polypeptide, or afunctional fragment or derivative thereof; determining a first level ofOPG expression and/or activity in said population of cells; eventuallyactivating the population of cells; exposing said population of cells toa compound, or a mixture of compounds; determining a second level of OPGexpression and/or activity in said population of cells during or afterexposure of said population of cells to said compound, or the mixture ofsaid compounds; and identifying the compound(s) that induce OPGexpression and/or activity.

As noted above, inhibition of osteoclastogenesis and bone resorption maybe determined by measuring the expression and/or activity of the TARGETpolypeptide and/or a known osteoclastogenesis and/or bone resorptioninhibiting protein. In a particular embodiment, said osteoclastogenesisand/or bone resorption inhibiting protein is able to prevent theformation of activated osteoclasts which act to remove bone tissue. In aspecific embodiment of the present invention, said osteoclastogenesisand/or bone resorption inhibiting protein is osteoprotegerin (OPG).

The expression of an osteoclastogenesis and/or bone resorptioninhibiting protein can be determined by methods known in the art such asWestern blotting using specific antibodies, or an ELISA using antibodiesspecifically recognizing a particular osteoclastogenesis and/or boneresorption inhibiting protein.

The present inventors have developed a protocol allowing the detection,in a high throughput mode, of the level of OPG in complex media such asthe supernatant of cultured cells.

The present inventors have developed a protocol allowing the detection,in a high throughput mode, of the level of osteoclast differentiation incomplex media and in monoculture or coculture, based on a cell-basedELISA for α_(v)β₃ integrin.

The present inventors identified TARGET genes involved inosteoclastogenesis and/or bone resorption by using a ‘knock-down’library. This type of library is a screen in which siRNA molecules aretransduced into cells by recombinant adenoviruses, which siRNA moleculesinhibit or repress the expression of a specific gene as well asexpression and activity of the corresponding gene product in a cell.Each siRNA in a viral vector corresponds to a specific natural gene. Byidentifying a siRNA that induces OPG expression, a direct correlationcan be drawn between the specific gene expression and the pathwaybetween OPG expression and activity and osteoclastogenesis leading tobone resorption. The TARGET genes identified using the knock-downlibrary (the protein expression products thereof herein referred to as“TARGET” polypeptides) are then used in the present inventive method foridentifying compounds that can be used to prevent bone resorption.Indeed, shRNA compounds comprising the sequences listed in Table 2(particularly SEQ ID NOs: 81-97 and 107, particularly SEQ ID NOs: 88,89, 91, 92 and 107) inhibit the expression and/or activity of theseTARGET genes and increase the OPG expression in cells, confirming therole of the TARGETS in the pathway from OPG expression to inhibition ofbone resorption.

TABLE 2 Exemplary KD target sequences useful in  the practice of the present expression- inhibitory agent inventionTARGET SEQ ID HIT ID SYMBOL TARGET KD Sequence NO: H51-082 NTRK2ATGCAGTGCCTCTCGGATC  81 H51-054 MAP4K4 TGGCACCTATGGACAAGTC  82 H51-104MMP17 CTGTTTGCAGTGGCTGTCC  83 H51-172 PLA2G12A TGCAGTGACGGATCTAAGC  84H51-181 MGLL CATGTTCTCCACAAGGAGC  85 H51-225 GPR44 CATGTTCGCCAGCGGCTTC 86 H51-236 MIR16 GTGGTCAGCTAAAGGAATC  87 H51-240 PTK6GAAGCTGCGGCACAAACAC  88 H51-137 MRAS AGAAATGGCGACCAAACAC  89 H51-121SLC4A8 AGCATGAGGGTTAAAGTGC  90 H51-122 ENPP2 CTGCAGTGCTTTATCGGAC  91H51-014 MAP3K3 TTCCTTGTCTGGAAGCTGC  92 H51-018 P2RY14GATCCTTGGTGACTCAGGC  93 H51-041 P2RY14 AGCTCAGAATGACCTAGAC  94 H51-040NEK3 GCAGTGGCTCAAAGAGACC  95 H51-046 KLKB1 CATCTGCACCTATCACCCC  96H51-142 FNTA/ TGGCTAAGAGATCCATCTC  97 LOC283226 H51-103 USP9YATGAACTCTGTGATCCAGC  98 H51-119 CDC7 TTCAGTGCCTAACAGTGGC  99 H51-145PPIA GCATGAATATTGTGGAGGC 100 H51-153 TOP2B AGCATGATGATAGTTCCTC 101H51-177 PPP2CB TGTGCAAGAGGTTCGTTGC 102 H51-183 COX10 TGCATGATGTCGGTCACCC103 H51-206 CCR1 AGCCTACGAGAGTGGAAGC 104 H51-251 B3GALT1AGTTTGTGTAGGTATCGCC 105 H51-270 SLC9A8 TGTTCTTTGGCTCTGCAGC 106 H51-261CXCR6 CTTCTACACGTCCATGCTC 107

Table 1 lists the TARGETS identified using applicants' knock-downlibrary in the OPG assay described below, including the class ofpolypeptides identified. TARGETS have been identified in polypeptideclasses including kinase, protease, enzyme, ion channel, GPCR,phosphodiesterase and phosphatase, for instance. Specific methods todetermine the activity of a kinase by measuring the phosphorylation of asubstrate by the kinase, which measurements are performed in thepresence or absence of a compound, are well known in the art.

Ion channels are membrane protein complexes and their function is tofacilitate the diffusion of ions across biological membranes. Membranes,or phospholipid bilayers, build a hydrophobic, low dielectric barrier tohydrophilic and charged molecules. Ion channels provide a highconducting, hydrophilic pathway across the hydrophobic interior of themembrane. The activity of an ion channel can be measured using classicalpatch clamping. High-throughput fluorescence-based or tracer-basedassays are also widely available to measure ion channel activity. Thesefluorescent-based assays screen compounds on the basis of their abilityto either open or close an ion channel thereby changing theconcentration of specific fluorescent dyes across a membrane. In thecase of the tracer-based assay, the changes in concentration of thetracer within and outside the cell are measured by radioactivitymeasurement or gas absorption spectrometry.

Specific methods to determine the inhibition by a compound by measuringthe cleavage of the substrate by the polypeptide, which is a protease,are well known in the art. Classically, substrates are used in which afluorescent group is linked to a quencher through a peptide sequencethat is a substrate that can be cleaved by the target protease. Cleavageof the linker separates the fluorescent group and quencher, giving riseto an increase in fluorescence.

G-protein coupled receptors (GPCR) are capable of activating an effectorprotein, resulting in changes in second messenger levels in the cell.The activity of a GPCR can be measured by measuring the activity levelof such second messengers. Two important and useful second messengers inthe cell are cyclic AMP (cAMP) and Ca²⁺. The activity levels can bemeasured by methods known to persons skilled in the art, either directlyby ELISA or radioactive technologies or by using substrates thatgenerate a fluorescent or luminescent signal when contacted with Ca²⁺ orindirectly by reporter gene analysis. The activity level of the one ormore secondary messengers may typically be determined with a reportergene controlled by a promoter, wherein the promoter is responsive to thesecond messenger. Promoters known and used in the art for such purposesare the cyclic-AMP responsive promoter that is responsive for thecyclic-AMP levels in the cell, and the NF-AT responsive promoter that issensitive to cytoplasmic Ca²⁺-levels in the cell. The reporter genetypically has a gene product that is easily detectable. The reportergene can either be stably infected or transiently transfected in thehost cell. Useful reporter genes are alkaline phosphatase, enhancedgreen fluorescent protein, destabilized green fluorescent protein,luciferase and β-galactosidase.

It should be understood that the cells expressing the polypeptides, maybe cells naturally expressing the polypeptides, or the cells may betransfected to express the polypeptides, as described above. Also, thecells may be transduced to overexpress the polypeptide, or may betransfected to express a non-endogenous form of the polypeptide, whichcan be differentially assayed or assessed.

In one particular embodiment the methods of the present inventionfurther comprise the step of contacting the population of cells with anagonist of the polypeptide. This is useful in methods wherein theexpression of the polypeptide in a certain chosen population of cells istoo low for a proper detection of its activity. By using an agonist thepolypeptide may be triggered, enabling a proper read-out if the compoundinhibits the polypeptide. Similar considerations apply to themeasurement of bone resorption. In a particular embodiment, the cellsused in the present method are mammalian synovial fibroblasts Thefibroblasts, in the assay contemplated, may be activated (e.g. bycytokines).

A method for identifying a compound that inhibits bone resorption,comprising:

-   -   (a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        41-69 and 80, and fragments thereof; and    -   (b) measuring a compound-polypeptide property related to bone        resorption.

In one embodiment of the present invention the compound-polypeptideproperty related to bone resorption is binding affinity.

In one embodiment of the present invention the compound-polypeptideproperty related to bone resorption is upregulation of a biologicalpathway producing a biochemical marker indicative of the inhibition ofbone resorption. In particular, in one embodiment the compound inducesor upregulates OPG activity or expression.

In one embodiment of the present invention the compound-polypeptideproperty related to bone resorption is the activity of said polypeptide.In particular, in one embodiment the compound inhibits the activity ofsaid polypeptide.

In one embodiment of the present invention the compound-polypeptideproperty related to bone resorption is the expression of saidpolypeptide. In particular, in one embodiment the compound inhibits theexpression of said polypeptide.

The present invention further relates to a method for identifying acompound that inhibits bone resorption, comprising:

-   -   a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        41-69 and 80;    -   b) determining the binding affinity of the compound to the        polypeptide;    -   c) contacting a population of mammalian cells expressing said        polypeptide with the compound that exhibits a binding affinity        of at least 10 micromolar; and    -   d) identifying the compound that inhibits bone resorption.

The present invention further relates to a method for identifying acompound that inhibits bone resorption, comprising:

-   -   a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        41-69 and 80;    -   b) determining the ability of the compound inhibit the        expression or activity of the polypeptide;    -   c) contacting a population of mammalian cells expressing said        polypeptide with the compound that significantly inhibits the        expression or activity of the polypeptide; and    -   d) identifying the compound that inhibits bone resorption.

In a particular aspect of the present invention the methods describedabove include the additional step of comparing the compound to be testedto a control, where the control is a population of cells that have notbeen contacted with the test compound.

In a particular aspect of the present invention the methods describedabove include the additional step of comparing the compound to be testedto a control, where the control is a population of cells that do notexpress said polypeptide.

The methods of the present invention may be performed in the presenceof, or in combination with, a Disease-Modifying Anti-Rheumatic Drug(DMARD), or an anti-inflammatory compound. The population of cells maybe exposed to the compound or the mixture of compounds through differentmeans, for instance by direct incubation in the medium, or by nucleicacid transfer into the cells. Such transfer may be achieved by a widevariety of means, for instance by direct transfection of naked isolatedDNA, or RNA, or by means of delivery systems, such as recombinantvectors. Other delivery means such as liposomes, or other lipid-basedvectors may also be used. Particularly, the nucleic acid compound isdelivered by means of a (recombinant) vector such as a recombinantvirus.

For high-throughput purposes, libraries of compounds may be used such asantibody fragment libraries, peptide phage display libraries, peptidelibraries (for example, LOPAP™, Sigma Aldrich), lipid libraries(BioMol), synthetic compound libraries (for example, LOPAC™, SigmaAldrich; BioFocus DPI) or natural compound libraries (Specs, TimTec).

Particular drug candidate compounds are low molecular weight compounds.Low molecular weight compounds, for example with a molecular weight of500 Dalton or less, are likely to have good absorption and permeation inbiological systems and are consequently more likely to be successfuldrug candidates than compounds with a molecular weight above 500 Dalton(Lipinski et al., (1997)). Peptides comprise another particular class ofdrug candidate compounds. Peptides may be excellent drug candidates andthere are multiple examples of commercially valuable peptides such asfertility hormones and platelet aggregation inhibitors. Naturalcompounds are another particular class of drug candidate compound. Suchcompounds are found in and extracted from natural sources, and which maythereafter be synthesized. The lipids are another particular class ofdrug candidate compound.

Another particular class of drug candidate compounds is an antibody. Thepresent invention also provides antibodies directed against a TARGET.These antibodies may be endogenously produced to bind to the TARGETwithin the cell, or added to the tissue to bind to TARGET polypeptidepresent outside the cell. These antibodies may be monoclonal antibodiesor polyclonal antibodies. The present invention includes chimeric,single chain, and humanized antibodies, as well as Fab fragments and theproducts of a Fab expression library, and Fv fragments and the productsof an Fv expression library. In another embodiment, the compound may bea nanobody, the smallest functional fragment of naturally occurringsingle-domain antibodies (Cortez-Retamozo et al. 2004).

In certain embodiments, polyclonal antibodies may be used in thepractice of the invention. The skilled artisan knows methods ofpreparing polyclonal antibodies. Polyclonal antibodies can be raised ina mammal, for example, by one or more injections of an immunizing agentand, if desired, an adjuvant. Typically, the immunizing agent and/oradjuvant will be injected in the mammal by multiple subcutaneous orintraperitoneal injections. Antibodies may also be generated against theintact TARGET protein or polypeptide, or against a fragment, derivativesincluding conjugates, or other epitope of the TARGET protein orpolypeptide, such as the TARGET embedded in a cellular membrane, or alibrary of antibody variable regions, such as a phage display library.

It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants that may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). One skilled in the art withoutundue experimentation may select the immunization protocol.

In some embodiments, the antibodies may be monoclonal antibodies.Monoclonal antibodies may be prepared using methods known in the art.The monoclonal antibodies of the present invention may be “humanized” toprevent the host from mounting an immune response to the antibodies. A“humanized antibody” is one in which the complementarity determiningregions (CDRs) and/or other portions of the light and/or heavy variabledomain framework are derived from a non-human immunoglobulin, but theremaining portions of the molecule are derived from one or more humanimmunoglobulins. Humanized antibodies also include antibodiescharacterized by a humanized heavy chain associated with a donor oracceptor unmodified light chain or a chimeric light chain, or viceversa. The humanization of antibodies may be accomplished by methodsknown in the art (see, for example, Mark and Padlan, (1994) “Chapter 4.Humanization of Monoclonal Antibodies”, The Handbook of ExperimentalPharmacology Vol. 113, Springer-Verlag, New York). Transgenic animalsmay be used to express humanized antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter,(1991) J. Mol. Biol. 227:381-8; Marks et al. (1991). J. Mol. Biol.222:581-97). The techniques of Cole, et al. and Boerner, et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole, etal. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p. 77;Boerner, et al (1991). J. Immunol., 147(1):86-95).

Techniques known in the art for the production of single chainantibodies can be adapted to produce single chain antibodies to theTARGET polypeptides and proteins of the present invention. Theantibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain cross-linking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent cross-linking

Bispecific antibodies are monoclonal, particularly human or humanized,antibodies that have binding specificities for at least two differentantigens and particularly for a cell-surface protein or receptor orreceptor subunit. In the present case, one of the binding specificitiesis for one domain of the TARGET, while the other one is for anotherdomain of the same or different TARGET.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, (1983) Nature 305:537-9). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. Affinitychromatography steps usually accomplish the purification of the correctmolecule. Similar procedures are disclosed in Trauneeker, et al. (1991)EMBO J. 10:3655-9.

According to another particular embodiment, the assay method uses a drugcandidate compound identified as having a binding affinity for a TARGET,and/or has already been identified as having down-regulating activitysuch as antagonist activity vis-à-vis one or more TARGET.

The present invention further relates to a method for inhibiting boneresorption comprising contacting mammalian cells with an expressioninhibitory agent comprising a polyribonucleotide sequence thatcomplements at least about 15 to about 30, particularly at least 17 toabout 30, most particularly at least 17 to about 25 contiguousnucleotides of a nucleotide sequence encoding a polypeptide TARGET orportion thereof, including the nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1-29 and 40, particularly selected fromthe group consisting of SEQ ID NO: 16, 17, 19-21 and 40.

Another aspect of the present invention relates to a method forinhibiting bone resorption, comprising by contacting mammalian cellswith an expression-inhibiting agent that inhibits the translation in thecell of a polyribonucleotide encoding a TARGET polypeptide. A particularembodiment relates to a composition comprising a polynucleotideincluding at least one antisense strand that functions to pair the agentwith the TARGET mRNA, and thereby down-regulate or block the expressionof TARGET polypeptide. The inhibitory agent particularly comprisesantisense polynucleotide, a ribozyme, and a small interfering RNA(siRNA), wherein said agent comprises a nucleic acid sequencecomplementary to, or engineered from, a naturally-occurringpolynucleotide sequence selected from the group consisting of SEQ ID NO:1-29 and 40, particularly selected from the group consisting of SEQ IDNO: 16, 17, 19-21 and 40.

A particular embodiment of the present invention relates to a methodwherein the expression-inhibiting agent is selected from the groupconsisting of antisense RNA, antisense oligodeoxynucleotide (ODN), aribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 41-69and 80, a small interfering RNA (siRNA, particularly shRNA,) that issufficiently homologous to a portion of the polyribonucleotidecorresponding to SEQ ID NO: 1-29 and 40, particularly selected from thegroup consisting of SEQ ID NO: 16, 17, 19-21 and 40, such that thesiRNA, particularly shRNA, interferes with the translation of the TARGETpolyribonucleotide to the TARGET polypeptide.

Another embodiment of the present invention relates to a method whereinthe expression-inhibiting agent is a nucleic acid expressing theantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide corresponding to SEQ ID NO: 1-29 and 40,particularly selected from the group consisting of SEQ ID NO: 16, 17,19-21 and 40, a small interfering RNA (siRNA, particularly shRNA,) thatis sufficiently complementary to a portion of the polyribonucleotidecorresponding to SEQ ID NO: 1-29 and 40, particularly selected from thegroup consisting of SEQ ID NO: 16, 17, 19-21 and 40, such that thesiRNA, particularly shRNA, interferes with the translation of the TARGETpolyribonucleotide to the TARGET polypeptide. Particularly theexpression-inhibiting agent is an antisense RNA, ribozyme, antisenseoligodeoxynucleotide, or siRNA, particularly shRNA, comprising apolyribonucleotide sequence that complements at least about 17 to about30 contiguous nucleotides of a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1-29 and 40, particularly selected fromthe group consisting of SEQ ID NO: 16, 17, 19-21 and 40. Moreparticularly, the expression-inhibiting agent is an antisense RNA,ribozyme, antisense oligodeoxynucleotide, or siRNA, particularly shRNA,comprising a polyribonucleotide sequence that complements at least 15 toabout 30, particularly at least 17 to about 30, most particularly atleast 17 to about 25 contiguous nucleotides of a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1-29 and 40,particularly selected from the group consisting of SEQ ID NO: 16, 17,19-21 and 40. A particular embodiment comprises a polyribonucleotidesequence that complements a polynucleotide sequence selected from thegroup consisting of SEQ ID NO: 81-97 and 107. A particular embodimentcomprises a polyribonucleotide sequence that complements apolynucleotide sequence selected from the group consisting of 88, 89,91, 92 and 107.

The down regulation of gene expression using antisense nucleic acids canbe achieved at the translational or transcriptional level. Antisensenucleic acids of the invention are particularly nucleic acid fragmentscapable of specifically hybridizing with all or part of a nucleic acidencoding a TARGET polypeptide or the corresponding messenger RNA. Inaddition, antisense nucleic acids may be designed which decreaseexpression of the nucleic acid sequence capable of encoding a TARGETpolypeptide by inhibiting splicing of its primary transcript. Any lengthof antisense sequence is suitable for practice of the invention so longas it is capable of down-regulating or blocking expression of a nucleicacid coding for a TARGET. Particularly, the antisense sequence is atleast about 15-30, and particularly at least 17 nucleotides in length.The preparation and use of antisense nucleic acids, DNA encodingantisense RNAs and the use of oligo and genetic antisense is known inthe art.

One embodiment of expression-inhibitory agent is a nucleic acid that isantisense to a nucleic acid comprising SEQ ID NO: 1-29 and 40,particularly SEQ ID NO: 16, 17, 19-21 and 40, for example, an antisensenucleic acid (for example, DNA) may be introduced into cells in vitro,or administered to a subject in vivo, as gene therapy to inhibitcellular expression of nucleic acids comprising SEQ ID NO: 1-29 and 40,particularly SEQ ID NO: 16, 17, 19-21 and 40. Antisense oligonucleotidesmay comprise a sequence containing from about 15 to about 100nucleotides, more particularly from about 15 to about 30 nucleotides,and most particularly, from about 17 to about 25 nucleotides. Antisensenucleic acids may be prepared from about 15 to about 30 contiguousnucleotides selected from the sequences of SEQ ID NO: 1-29 and 40,particularly SEQ ID NO: 16, 17, 19-21 and 40, expressed in the oppositeorientation.

The skilled artisan can readily utilize any of several strategies tofacilitate and simplify the selection process for antisense nucleicacids and oligonucleotides effective in inhibition of TARGET and/or OPGexpression. Predictions of the binding energy or calculation ofthermodynamic indices between an olionucleotide and a complementarysequence in an mRNA molecule may be utilized (Chiang et al. (1991) J.Biol. Chem. 266:18162-18171; Stull et al. (1992) Nucl. Acids Res.20:3501-3508). Antisense oligonucleotides may be selected on the basisof secondary structure (Wickstrom et al (1991) in Prospects forAntisense Nucleic Acid Therapy of Cancer and AIDS, Wickstrom, ed.,Wiley-Liss, Inc., New York, pp. 7-24; Lima et al. (1992) Biochem.31:12055-12061). Schmidt and Thompson (U.S. Pat. No. 6,416,951) describea method for identifying a functional antisense agent comprisinghybridizing an RNA with an oligonucleotide and measuring in real timethe kinetics of hybridization by hybridizing in the presence of anintercalation dye or incorporating a label and measuring thespectroscopic properties of the dye or the label's signal in thepresence of unlabelled oligonucleotide. In addition, any of a variety ofcomputer programs may be utilized which predict suitable antisenseoligonucleotide sequences or antisense targets utilizing variouscriteria recognized by the skilled artisan, including for example theabsence of self-complementarity, the absence hairpin loops, the absenceof stable homodimer and duplex formation (stability being assessed bypredicted energy in kcal/mol). Examples of such computer programs arereadily available and known to the skilled artisan and include the OLIGO4 or OLIGO 6 program (Molecular Biology Insights, Inc., Cascade, Colo.)and the Oligo Tech program (Oligo Therapeutics Inc., Wilsonville,Oreg.). In addition, antisense oligonucleotides suitable in the presentinvention may be identified by screening an oligonucleotide library, ora library of nucleic acid molecules, under hybridization conditions andselecting for those which hybridize to the target RNA or nucleic acid(see for example U.S. Pat. No. 6,500,615). Mishra and Toulme have alsodeveloped a selection procedure based on selective amplification ofoligonucleotides that bind target (Mishra et al (1994) Life Sciences317:977-982). Oligonucleotides may also be selected by their ability tomediate cleavage of target RNA by RNAse H, by selection andcharacterization of the cleavage fragments (Ho et al (1996) Nucl AcidsRes 24:1901-1907; Ho et al (1998) Nature Biotechnology 16:59-630).Generation and targeting of oligonucleotides to GGGA motifs of RNAmolecules has also been described (U.S. Pat. No. 6,277,981).

The antisense nucleic acids are particularly oligonucleotides and mayconsist entirely of deoxyribo-nucleotides, modifieddeoxyribonucleotides, or some combination of both. The antisense nucleicacids can be synthetic oligonucleotides. The oligonucleotides may bechemically modified, if desired, to improve stability and/orselectivity. Specific examples of some particular oligonucleotidesenvisioned for this invention include those containing modifiedbackbones, for example, phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar linkages. Sinceoligonucleotides are susceptible to degradation by intracellularnucleases, the modifications can include, for example, the use of asulfur group to replace the free oxygen of the phosphodiester bond. Thismodification is called a phosphorothioate linkage. Phosphorothioateantisense oligonucleotides are water soluble, polyanionic, and resistantto endogenous nucleases. In addition, when a phosphorothioate antisenseoligonucleotide hybridizes to its TARGET site, the RNA-DNA duplexactivates the endogenous enzyme ribonuclease (RNase) H, which cleavesthe mRNA component of the hybrid molecule. Oligonucleotides may alsocontain one or more substituted sugar moieties. Particularoligonucleotides comprise one of the following at the 2′ position: OH,SH, SCH₃, F, OCN, heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino; substituted silyl; an RNA cleaving group; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. Similar modifications may also be made atother positions on the oligonucleotide, particularly the 3′ position ofthe sugar on the 3′ terminal nucleotide and the 5′ position of 5′terminal nucleotide.

In addition, antisense oligonucleotides with phosphoramidite andpolyamide (peptide) linkages can be synthesized. These molecules shouldbe very resistant to nuclease degradation. Furthermore, chemical groupscan be added to the 2′ carbon of the sugar moiety and the 5 carbon (C-5)of pyrimidines to enhance stability and facilitate the binding of theantisense oligonucleotide to its TARGET site. Modifications may include2′-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxyphosphorothioates, modified bases, as well as other modifications knownto those of skill in the art.

Another type of expression-inhibitory agent that reduces the levels ofTARGETS is the ribozyme. Ribozymes are catalytic RNA molecules (RNAenzymes) that have separate catalytic and substrate binding domains Thesubstrate binding sequence combines by nucleotide complementarity and,possibly, non-hydrogen bond interactions with its TARGET sequence. Thecatalytic portion cleaves the TARGET RNA at a specific site. Thesubstrate domain of a ribozyme can be engineered to direct it to aspecified mRNA sequence. The ribozyme recognizes and then binds a TARGETmRNA through complementary base pairing. Once it is bound to the correctTARGET site, the ribozyme acts enzymatically to cut the TARGET mRNA.Cleavage of the mRNA by a ribozyme destroys its ability to directsynthesis of the corresponding polypeptide. Once the ribozyme hascleaved its TARGET sequence, it is released and can repeatedly bind andcleave at other mRNAs.

Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitisdelta virus, group I intron or RNaseP RNA (in association with an RNAguide sequence) motif or Neurospora VS RNA motif. Ribozymes possessing ahammerhead or hairpin structure are readily prepared since thesecatalytic RNA molecules can be expressed within cells from eukaryoticpromoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9). A ribozymeof the present invention can be expressed in eukaryotic cells from theappropriate DNA vector. If desired, the activity of the ribozyme may beaugmented by its release from the primary transcript by a secondribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21:3249-55).

Ribozymes may be chemically synthesized by combining anoligodeoxyribonucleotide with a ribozyme catalytic domain (20nucleotides) flanked by sequences that hybridize to the TARGET mRNAafter transcription. The oligodeoxyribonucleotide is amplified by usingthe substrate binding sequences as primers. The amplification product iscloned into a eukaryotic expression vector.

Ribozymes are expressed from transcription units inserted into DNA, RNA,or viral vectors. Transcription of the ribozyme sequences are drivenfrom a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymeraseII (pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters will be expressed at high levels in all cells; thelevels of a given pol II promoter in a given cell type will depend onnearby gene regulatory sequences. Prokaryotic RNA polymerase promotersare also used, providing that the prokaryotic RNA polymerase enzyme isexpressed in the appropriate cells (Gao and Huang, (1993) Nucleic AcidsRes. 21:2867-72). It has been demonstrated that ribozymes expressed fromthese promoters can function in mammalian cells (Kashani-Sabet, et al.(1992) Antisense Res. Dev. 2:3-15).

A particular inhibitory agent is a small interfering RNA (siRNA,particularly small hairpin RNA, “shRNA”). siRNA, particularly shRNA,mediate the post-transcriptional process of gene silencing by doublestranded RNA (dsRNA) that is homologous in sequence to the silenced RNA.siRNA according to the present invention comprises a sense strand of15-30, particularly 17-30, most particularly 17-25 nucleotidescomplementary or homologous to a contiguous 17-25 nucleotide sequenceselected from the group of sequences described in SEQ ID NO: 1-29 and40, particularly SEQ ID NO: 16, 17, 19-21 and 40, particularly from thegroup of sequences described in SEQ ID No: 81-97 and 107, mostparticularly those described in SEQ ID NO: 88, 89, 91, 92 and 107, andan antisense strand of 15-30, particularly 17-30, most particularly17-25 nucleotides complementary to the sense strand. The most particularsiRNA comprises sense and anti-sense strands that are 100 percentcomplementary to each other and the TARGET polynucleotide sequence.Particularly the siRNA further comprises a loop region linking the senseand the antisense strand.

A self-complementing single stranded shRNA molecule polynucleotideaccording to the present invention comprises a sense portion and anantisense portion connected by a loop region linker. Particularly, theloop region sequence is 4-30 nucleotides long, more particularly 5-15nucleotides long and most particularly 8 or 12 nucleotides long. In amost particular embodiment the linker sequence is UUGCUAUA orGUUUGCUAUAAC (SEQ ID NO: 108). Self-complementary single stranded siRNAsform hairpin loops and are more stable than ordinary dsRNA. In addition,they are more easily produced from vectors.

Analogous to antisense RNA, the siRNA can be modified to conferresistance to nucleolytic degradation, or to enhance activity, or toenhance cellular distribution, or to enhance cellular uptake, suchmodifications may consist of modified internucleoside linkages, modifiednucleic acid bases, modified sugars and/or chemical linkage the siRNA toone or more moieties or conjugates. The nucleotide sequences may beselected according to siRNA designing rules that give an improvedreduction of the TARGET sequences compared to nucleotide sequences thatdo not comply with these siRNA designing rules (For a discussion ofthese rules and examples of the preparation of siRNA, WO 2004/094636 andUS 2003/0198627, are hereby incorporated by reference).

The present invention also relates to compositions, and methods usingsaid compositions, comprising a DNA expression vector capable ofexpressing a polynucleotide capable of inhibiting bone resorption anddescribed hereinabove as an expression inhibition agent.

A particular aspect of these compositions and methods relates to thedown-regulation or blocking of the expression of a TARGET polypeptide bythe induced expression of a polynucleotide encoding an intracellularbinding protein that is capable of selectively interacting with theTARGET polypeptide. An intracellular binding protein includes anyprotein capable of selectively interacting, or binding, with thepolypeptide in the cell in which it is expressed and neutralizing orotherwise inhibiting or blocking the function of the polypeptide.Particularly, the intracellular binding protein is a neutralizingantibody or a fragment of a neutralizing antibody having bindingaffinity to an epitope of the TARGET polypeptide of SEQ ID NO: 41-69 and80, particularly to an epitope of the TARGET polypeptide of SEQ ID NO:56, 57, 59-61 and 80. More particularly, the intracellular bindingprotein is a single chain antibody.

A particular embodiment of this composition comprises theexpression-inhibiting agent selected from the group consisting ofantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for SEQ ID NO: 41-69 or 80,particularly SEQ ID NO: 56, 57, 59-61 or 80, and a small interfering RNA(siRNA) that is sufficiently homologous to a portion of thepolyribonucleotide corresponding to SEQ ID NO: 1-29 and 40, particularlySEQ ID NO: 16, 17, 19-21 and 40, such that the siRNA interferes with thetranslation of the TARGET polyribonucleotide to the TARGET polypeptide.

The polynucleotide expressing the expression-inhibiting agent, or apolynucleotide expressing the TARGET polypeptide in cells, isparticularly included within a vector. The polynucleic acid is operablylinked to signals enabling expression of the nucleic acid sequence andis introduced into a cell utilizing, particularly, recombinant vectorconstructs, which will express the nucleic acid or antisense nucleicacid once the vector is introduced into the cell. A variety ofviral-based systems are available, including adenoviral, retroviral,adeno-associated viral, lentiviral, herpes simplex viral or a sendaviralvector systems. All may be used to introduce and express polynucleotidesequence for the expression-inhibiting agents in TARGET cells.

Particularly, the viral vectors used in the methods of the presentinvention are replication defective. Such replication defective vectorswill usually pack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution, partial deletion or addition ofone or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Particularly, the replication defective virus retainsthe sequences of its genome, which are necessary for encapsidating, theviral particles.

In a particular embodiment, the viral element is derived from anadenovirus. Particularly, the vehicle includes an adenoviral vectorpackaged into an adenoviral capsid, or a functional part, derivative,and/or analogue thereof. Adenovirus biology is also comparatively wellknown on the molecular level. Many tools for adenoviral vectors havebeen and continue to be developed, thus making an adenoviral capsid aparticular vehicle for incorporating in a library of the invention. Anadenovirus is capable of infecting a wide variety of cells. However,different adenoviral serotypes have different preferences for cells. Tocombine and widen the TARGET cell population that an adenoviral capsidof the invention can enter in a particular embodiment, the vehicleincludes adenoviral fiber proteins from at least two adenoviruses.Particular adenoviral fiber protein sequences are serotype 17, 45 and51. Techniques or construction and expression of these chimeric vectorsare disclosed in US 2003/0180258 and US 2004/0071660, herebyincorporated by reference.

In a particular embodiment, the nucleic acid derived from an adenovirusincludes the nucleic acid encoding an adenoviral late protein or afunctional part, derivative, and/or analogue thereof. An adenoviral lateprotein, for instance an adenoviral fiber protein, may be favorably usedto TARGET the vehicle to a certain cell or to induce enhanced deliveryof the vehicle to the cell. Particularly, the nucleic acid derived froman adenovirus encodes for essentially all adenoviral late proteins,enabling the formation of entire adenoviral capsids or functional parts,analogues, and/or derivatives thereof. Particularly, the nucleic acidderived from an adenovirus includes the nucleic acid encoding adenovirusE2A or a functional part, derivative, and/or analogue thereof.Particularly, the nucleic acid derived from an adenovirus includes thenucleic acid encoding at least one E4-region protein or a functionalpart, derivative, and/or analogue thereof, which facilitates, at leastin part, replication of an adenoviral derived nucleic acid in a cell.The adenoviral vectors used in the examples of this application areexemplary of the vectors useful in the present method of treatmentinvention.

Certain embodiments of the present invention use retroviral vectorsystems. Retroviruses are integrating viruses that infect dividingcells, and their construction is known in the art. Retroviral vectorscan be constructed from different types of retrovirus, such as, MoMuLV(“Moloney murine leukemia virus” MSV (“Moloney murine sarcoma virus”),HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Roussarcoma virus”) and Friend virus. Lentiviral vector systems may also beused in the practice of the present invention. Retroviral systems andherpes virus system may be particular vehicles for transfection ofneuronal cells.

In other embodiments of the present invention, adeno-associated viruses(“AAV”) are utilized. The AAV viruses are DNA viruses of relativelysmall size that integrate, in a stable and site-specific manner, intothe genome of the infected cells. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies.

In the vector construction, the polynucleotide agents of the presentinvention may be linked to one or more regulatory regions. Selection ofthe appropriate regulatory region or regions is a routine matter, withinthe level of ordinary skill in the art. Regulatory regions includepromoters, and may include enhancers, suppressors, etc.

Promoters that may be used in the expression vectors of the presentinvention include both constitutive promoters and regulated (inducible)promoters. The promoters may be prokaryotic or eukaryotic depending onthe host. Among the prokaryotic (including bacteriophage) promotersuseful for practice of this invention are lac, lacZ, T3, T7, lambdaP_(r), P_(l), and trp promoters. Among the eukaryotic (including viral)promoters useful for practice of this invention are ubiquitous promoters(for example, HPRT, vimentin, actin, tubulin), intermediate filamentpromoters (for example, desmin, neurofilaments, keratin, GFAP),therapeutic gene promoters (for example, MDR type, CFTR, factor VIII),tissue-specific promoters (for example, actin promoter in smooth musclecells, or Flt and Flk promoters active in endothelial cells), includinganimal transcriptional control regions, which exhibit tissue specificityand have been utilized in transgenic animals: elastase I gene controlregion which is active in pancreatic acinar cells (Swift, et al. (1984)Cell 38:639-46; Ornitz, et al. (1986) Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, (1987) Hepatology 7:425-515); insulin genecontrol region which is active in pancreatic beta cells (Hanahan, (1985)Nature 315:115-22), immunoglobulin gene control region which is activein lymphoid cells (Grosschedl, et al. (1984) Cell 38:647-58; Adames, etal. (1985) Nature 318:533-8; Alexander, et al. (1987) Mol. Cell. Biol.7:1436-44), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder, et al. (1986) Cell45:485-95), albumin gene control region which is active in liver(Pinkert, et al. (1987) Genes and Devel. 1:268-76), alpha-fetoproteingene control region which is active in liver (Krumlauf, et al. (1985)Mol. Cell. Biol., 5:1639-48; Hammer, et al. (1987) Science 235:53-8),alpha 1-antitrypsin gene control region which is active in the liver(Kelsey, et al. (1987) Genes and Devel., 1: 161-71), beta-globin genecontrol region which is active in myeloid cells (Mogram, et al. (1985)Nature 315:338-40; Kollias, et al. (1986) Cell 46:89-94), myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead, et al. (1987) Cell 48:703-12), myosin light chain-2gene control region which is active in skeletal muscle (Sani, (1985)Nature 314.283-6), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason, et al. (1986) Science234:1372-8).

Other promoters which may be used in the practice of the inventioninclude promoters which are preferentially activated in dividing cells,promoters which respond to a stimulus (for example, steroid hormonereceptor, retinoic acid receptor), tetracycline-regulatedtranscriptional modulators, cytomegalovirus immediate-early, retroviralLTR, metallothionein, SV-40, E1a, and MLP promoters.

Additional vector systems include the non-viral systems that facilitateintroduction of polynucleotide agents into a patient, for example, a DNAvector encoding a desired sequence can be introduced in vivo bylipofection. Synthetic cationic lipids designed to limit thedifficulties encountered with liposome-mediated transfection can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner, et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7);see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer,et al. (1993) Science 259:1745-8). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold, (1989) Nature 337:387-8). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO 95/18863 and WO 96/17823, and inU.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenousgenes into the specific organs in vivo has certain practical advantagesand directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, forexample, pancreas, liver, kidney, and the brain. Lipids may bechemically coupled to other molecules for the purpose of targeting.Targeted peptides, for example, hormones or neurotransmitters, andproteins, for example, antibodies, or non-peptide molecules could becoupled to liposomes chemically. Other molecules are also useful forfacilitating transfection of a nucleic acid in vivo, for example, acationic oligopeptide (for example, International Patent Publication WO95/21931), peptides derived from DNA binding proteins (for example,International Patent Publication WO 96/25508), or a cationic polymer(for example, International Patent Publication WO 95/21931).

It is also possible to introduce a DNA vector in vivo as a naked DNAplasmid (see U.S. Pat. Nos. 5,693,622; 5,589,466; and 5,580,859). NakedDNA vectors for therapeutic purposes can be introduced into the desiredhost cells by methods known in the art, for example, transfection,electro-poration, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, for example, Wilson, et al. (1992) J. Biol.Chem. 267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut,et al. Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30).Receptor-mediated DNA delivery approaches can also be used (Curiel, etal. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem.262:4429-32).

The present invention also provides biologically compatible, boneresorption inhibiting compositions comprising an effective amount of oneor more compounds identified as TARGET inhibitors, and/or theexpression-inhibiting agents as described hereinabove.

A biologically compatible composition is a composition, that may besolid, liquid, gel, or other form, in which the compound,polynucleotide, vector, or antibody of the invention is maintained in anactive form, for example, in a form able to affect a biologicalactivity. For example, a compound of the invention would have inverseagonist or antagonist activity on the TARGET; a nucleic acid would beable to replicate, translate a message, or hybridize to a complementarymRNA of a TARGET; a vector would be able to transfect a TARGET cell andexpress the antisense, antibody, ribozyme or siRNA as describedhereinabove; an antibody would bind a TARGET polypeptide domain.

A particular biologically compatible composition is an aqueous solutionthat is buffered using, for example, Tris, phosphate, or HEPES buffer,containing salt ions. Usually the concentration of salt ions will besimilar to physiological levels. Biologically compatible solutions mayinclude stabilizing agents and preservatives. In a more particularembodiment, the biocompatible composition is a pharmaceuticallyacceptable composition. Such compositions can be formulated foradministration by topical, oral, parenteral, intranasal, subcutaneous,and intraocular, routes. Parenteral administration is meant to includeintravenous injection, intramuscular injection, intraarterial injectionor infusion techniques. The composition may be administered parenterallyin dosage unit formulations containing standard, well-known non-toxicphysiologically acceptable carriers, adjuvants and vehicles as desired.

A particular embodiment of the present composition invention is a boneresorption inhibiting pharmaceutical composition comprising atherapeutically effective amount of an expression-inhibiting agent asdescribed hereinabove, in admixture with a pharmaceutically acceptablecarrier. Another particular embodiment is a pharmaceutical compositionfor the treatment or prevention of a condition involving boneresorption, or a susceptibility to the condition, comprising aneffective bone resorption inhibiting amount of a TARGET antagonist orinverse agonist, its pharmaceutically acceptable salts, hydrates,solvates, or prodrugs thereof in admixture with a pharmaceuticallyacceptable carrier.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragées,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient. Pharmaceutical compositions for oral usecan be prepared by combining active compounds with solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethyl-cellulose; gums including arabic and tragacanth;and proteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate. Dragee cores may be used in conjunction with suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification or to characterizethe quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Particular sterile injectable preparations can be a solution orsuspension in a non-toxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, bufferedsaline, isotonic saline (for example, monosodium or disodium phosphate,sodium, potassium; calcium or magnesium chloride, or mixtures of suchsalts), Ringer's solution, dextrose, water, sterile water, glycerol,ethanol, and combinations thereof 1,3-butanediol and sterile fixed oilsare conveniently employed as solvents or suspending media. Any blandfixed oil can be employed including synthetic mono- or di-glycerides.Fatty acids such as oleic acid also find use in the preparation ofinjectables.

The compounds or compositions of the invention may be combined foradministration with or embedded in polymeric carrier(s), biodegradableor biomimetic matrices or in a scaffold. The carrier, matrix or scaffoldmay be of any material that will allow composition to be incorporatedand expressed and will be compatible with the addition of cells or inthe presence of cells. Particularly, the carrier matrix or scaffold ispredominantly non-immunogenic and is biodegradable. Examples ofbiodegradable materials include, but are not limited to, polyglycolicacid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suturematerial, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin,alginate, cotton, or other naturally-occurring biodegradable materials.It may be preferable to sterilize the matrix or scaffold material priorto administration or implantation, e.g., by treatment with ethyleneoxide or by gamma irradiation or irradiation with an electron beam. Inaddition, a number of other materials may be used to form the scaffoldor framework structure, including but not limited to: nylon(polyamides), dacron (polyesters), polystyrene, polypropylene,polyacrylates, polyvinyl compounds (e.g., polyvinylchloride),polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox(TPX), polymers of hydroxy acids such as polylactic acid (PLA),polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA),polyorthoesters, polyanhydrides, polyphosphazenes, and a variety ofpolyhydroxyalkanoates, and combinations thereof. Matrices suitableinclude a polymeric mesh or sponge and a polymeric hydrogel. In theparticular embodiment, the matrix is biodegradable over a time period ofless than a year, more particularly less than six months, mostparticularly over two to ten weeks. The polymer composition, as well asmethod of manufacture, can be used to determine the rate of degradation.For example, mixing increasing amounts of polylactic acid withpolyglycolic acid decreases the degradation time. Meshes of polyglycolicacid that can be used can be obtained commercially, for instance, fromsurgical supply companies (e.g., Ethicon, N.J). In general, thesepolymers are at least partially soluble in aqueous solutions, such aswater, buffered salt solutions, or aqueous alcohol solutions, that havecharged side groups, or a monovalent ionic salt thereof.

The composition medium can also be a hydrogel, which is prepared fromany biocompatible or non-cytotoxic homo- or hetero-polymer, such as ahydrophilic polyacrylic acid polymer that can act as a drug absorbingsponge. Certain of them, such as, in particular, those obtained fromethylene and/or propylene oxide are commercially available. A hydrogelcan be deposited directly onto the surface of the tissue to be treated,for example during surgical intervention.

Embodiments of pharmaceutical compositions of the present inventioncomprise a replication defective recombinant viral vector encoding theagent of the present invention and a transfection enhancer, such aspoloxamer. An example of a poloxamer is Poloxamer 407, which iscommercially available (BASF, Parsippany, N.J.) and is a non-toxic,biocompatible polyol. A poloxamer impregnated with recombinant virusesmay be deposited directly on the surface of the tissue to be treated,for example during a surgical intervention. Poloxamer possessesessentially the same advantages as hydrogel while having a lowerviscosity.

The active agents may also be entrapped in microcapsules prepared, forexample, by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980) 16th edition, Osol, A. Ed.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, for example, films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™. (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

As defined above, therapeutically effective dose means that amount ofcompound, agent, protein, polynucleotide, peptide, or its antibodies,agonists or antagonists, which ameliorate a condition or one or moresymptoms thereof. Therapeutic efficacy and toxicity of such compoundscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, for example, ED₅₀ (the dose therapeuticallyeffective in 50% of the population) and LD₅₀ (the dose lethal to 50% ofthe population). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.Pharmaceutical compositions that exhibit large therapeutic indices areparticular. The data obtained from cell culture assays and animalstudies are used in formulating a range of dosage for human use. Thedosage of such compounds lies particularly within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The pharmaceutical compositions according to this invention may beadministered to a subject by a variety of methods. They may be addeddirectly to targeted tissues, complexed with cationic lipids, packagedwithin liposomes, or delivered to targeted cells by other methods knownin the art. Localized administration to the desired tissues may be doneby direct injection, transdermal absorption, catheter, infusion pump orstent. The DNA, DNA/vehicle complexes, or the recombinant virusparticles are locally administered to the site of treatment. Alternativeroutes of delivery include, but are not limited to, intravenousinjection, intramuscular injection, subcutaneous injection, aerosolinhalation, oral (tablet or pill form), topical, systemic, ocular,intraperitoneal and/or intrathecal delivery. Examples of ribozymedelivery and administration are provided in Sullivan et al. WO 94/02595.

Antibodies according to the invention may be delivered as a bolus only,infused over time or both administered as a bolus and infused over time.Those skilled in the art may employ different formulations forpolynucleotides than for proteins. Similarly, delivery ofpolynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

As discussed hereinabove, recombinant viruses may be used to introduceDNA encoding polynucleotide agents useful in the present invention.Recombinant viruses according to the invention are generally formulatedand administered in the form of doses of between about 10⁴ and about10¹⁴ pfu. In the case of AAVs and adenoviruses, doses of from about 10⁶to about 10¹¹ pfu are particularly used. The term pfu (“plaque-formingunit”) corresponds to the infective power of a suspension of virions andis determined by infecting an appropriate cell culture and measuring thenumber of plaques formed. The techniques for determining the pfu titreof a viral solution are well documented in the prior art.

The present invention also provides methods of inhibiting bone or jointdegradation, comprising administering, to a subject suffering from adisease condition involving bone or joint degradation, a bone or jointdegradation inhibiting pharmaceutical composition as described herein,particularly a therapeutically effective amount of anexpression-inhibiting agent of the present invention.

The present invention further provides methods of reducing the number orprevalence of bone fractures, comprising administering, to a subjectsuffering from a disease condition involving bone or joint degradation,a bone or joint degradation inhibiting pharmaceutical composition asdescribed herein, particularly a therapeutically effective amount of anexpression-inhibiting agent of the present invention. The diseasesinvolving bone resorption, include osteoporosis, juvenile osteoporosis,osteogenesis imperfecta, hypercalcemia, hyperparathyroidism,osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis,Paget's disease of bone, bone loss due to rheumatoid arthritis,inflammatory arthritis, osteomyelitis, corticosteroid treatment,metastatic bone diseases, periodontal bone loss, bone loss due tocancer, age-related loss of bone mass, other forms of osteopenia. Moreparticular diseases for treatment in accordance with the presentinvention are the degenerative joint diseases such as rheumatoidarthritis, psoriatic arthritis, juvenile arthritis, early arthritis,reactive arthritis, osteoarthritis, ankylosing spondylitis. The mostparticular degenerative joint disease for treatment in accordance withthe present method is rheumatoid arthritis.

The present invention also provides methods of inhibiting bone or jointdegradation, comprising administering, to a subject suffering from adisease condition involving bone or joint degradation, a bone resorptioninhibiting pharmaceutical composition as described herein, particularlya therapeutically effective amount of an agent which inhibits theexpression or activity of a TARGET as identified herein. The diseasesinvolving bone or joint degradation, include osteoporosis, juvenileosteoporosis, osteogenesis imperfecta, hypercalcemia,hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bonedisease, osteonecrosis, Paget's disease of bone, bone loss due torheumatoid arthritis, inflammatory arthritis, osteomyelitis,corticosteroid treatment, metastatic bone diseases, periodontal boneloss, bone loss due to cancer, age-related loss of bone mass, otherforms of osteopenia. More particular diseases for treatment inaccordance with the present invention are the degenerative jointdiseases such as rheumatoid arthritis, psoriatic arthritis, juvenilearthritis, early arthritis, reactive arthritis, osteoarthritis,ankylosing spondylitis. The most particular degenerative joint diseasefor treatment in accordance with the present method is rheumatoidarthritis.

In a further aspect the present invention provides methods of inhibitingbone or joint degradation, comprising administering, to a subjectsuffering from a disease condition involving bone or joint degradation,a bone resorption inhibiting pharmaceutical composition as describedherein, particularly a therapeutically effective amount of an agentwhich inhibits the expression or activity of a TARGET as identifiedherein in combination with a disease-modifying anti-rheumatic drug(DMARD) or an anti-inflammatory compound. The diseases involving bone orjoint degradation, include osteoporosis, juvenile osteoporosis,osteogenesis imperfecta, hypercalcemia, hyperparathyroidism,osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis,Paget's disease of bone, bone loss due to rheumatoid arthritis,inflammatory arthritis, osteomyelitis, corticosteroid treatment,metastatic bone diseases, periodontal bone loss, bone loss due tocancer, age-related loss of bone mass, other forms of osteopenia.Particular anti-inflammatory compounds include corticosteroids ornon-steroidal anti-inflammatory agents. Particular DMARDs includebiological DMARDs such as Infliximab, Etanercept, Adalimumab, Rituximabor CTLA4-Ig or synthetic DMARDs such as methotrexate, leflunomide orsulfasalazine. More particular diseases for treatment in accordance withthe present invention are the degenerative joint diseases such asrheumatoid arthritis, psoriatic arthritis, juvenile arthritis, earlyarthritis, reactive arthritis, osteoarthritis, ankylosing spondylitis.The most particular degenerative joint disease for treatment inaccordance with the present method is rheumatoid arthritis.

Administration of the expression-inhibiting agent of the presentinvention to the subject patient includes both self-administration andadministration by another person. The patient may be in need oftreatment for an existing disease or medical condition, or may desireprophylactic treatment to prevent or reduce the risk for diseases andmedical conditions affected by a disturbance in bone metabolism. Theexpression-inhibiting agent of the present invention may be delivered tothe subject patient orally, transdermally, via inhalation, injection,nasally, rectally or via a sustained release formulation.

A particular regimen of the present method comprises the administrationto a subject suffering from a disease condition characterized by adisturbance in bone metabolism, an effective bone resorption inhibitingamount of an expression-inhibiting agent of the present invention for aperiod of time sufficient to reduce the abnormal levels of boneresorption in the patient, and particularly terminate, theself-perpetuating processes responsible for said resorption. Aparticular embodiment of the method comprises administering of aneffective OPG inducing amount of a expression-inhibiting agent of thepresent invention to a subject patient suffering from or susceptible tothe development of rheumatoid arthritis, for a period of time sufficientto reduce or prevent, respectively, bone resorption in the joints ofsaid patient, and particularly terminate, the self-perpetuatingprocesses responsible for said resorption.

The invention also relates to the use of an agent as described above forthe preparation of a medicament for treating or preventing a diseaseinvolving bone resorption. Particularly the pathological condition isarthritis. More particularly, the pathological condition is rheumatoidarthritis.

The polypeptides and polynucleotides useful in the practice of thepresent invention described herein may be free in solution, affixed to asolid support, borne on a cell surface, or located intracellularly. Toperform the methods it is feasible to immobilize either the TARGETpolypeptide or the compound to facilitate separation of complexes fromuncomplexed forms of the polypeptide, as well as to accommodateautomation of the assay. Interaction (for example, binding of) of theTARGET polypeptide with a compound can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotitre plates, test tubes, and microcentrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows the polypeptide to be bound to a matrix. For example, the TARGETpolypeptide can be “His” tagged, and subsequently adsorbed onto Ni-NTAmicrotitre plates, or ProtA fusions with the TARGET polypeptides can beadsorbed to IgG, which are then combined with the cell lysates (forexample, ³⁵S-labelled) and the candidate compound, and the mixtureincubated under conditions favorable for complex formation (for example,at physiological conditions for salt and pH). Following incubation, theplates are washed to remove any unbound label, and the matrix isimmobilized. The amount of radioactivity can be determined directly, orin the supernatant after dissociation of the complexes. Alternatively,the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of the protein binding to the TARGET proteinquantified from the gel using standard electro-phoretic techniques.

Other techniques for immobilizing protein on matrices can also be usedin the method of identifying compounds. For example, either the TARGETor the compound can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated TARGET protein molecules can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques well known in the art(for example, biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with the TARGETS but whichdo not interfere with binding of the TARGET to the compound can bederivatized to the wells of the plate, and the TARGET can be trapped inthe wells by antibody conjugation. As described above, preparations of alabeled candidate compound are incubated in the wells of the platepresenting the TARGETS, and the amount of complex trapped in the wellcan be quantitated.

The polynucleotides encoding the TARGET polypeptides are identified asSEQ ID NO: 1-29 and 40. The present inventors show herein thattransfection of mammalian cells with Ad-siRNAs targeting these genesdecreases the release of factors that promote osteoclast differentiationand bone resorption.

The present invention also relates to a method for diagnosis of apathological condition involving bone resorption, comprising determiningthe nucleic acid sequence of at least one of the genes of SEQ ID NO:1-29 and 40, particularly SEQ ID NO: 16, 17, 19-21 and 40 within thegenomic DNA of a subject; comparing the sequence with the nucleic acidsequence obtained from a database and/or a healthy subject; andidentifying any difference(s) related to the onset of the pathologicalcondition.

Still another aspect of the invention relates to a method for diagnosinga pathological condition involving bone resorption or a susceptibilityto the condition in a subject, comprising determining the amount ofpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 41-69 and 80, particularly SEQ ID NO: 56, 57,59-61 and 80 in a biological sample, and comparing the amount with theamount of the polypeptide in a healthy subject, wherein an increase ofthe amount of polypeptide compared to the healthy subject is indicativeof the presence of the pathological condition.

The invention is further illustrated in the following figures andexamples.

EXAMPLES

As described in the introduction, OPG has been identified in thearthritis and osteoporosis literature as one of the key players involvedin the regulation of the differentiation and activity of osteoclasts andtherefore of the maintenance of bone. Current bone protective therapiesdisplay a lack of efficacy in rheumatoid arthritis. Increasedconcentration of RANKL, originating from synovial fibroblasts andT-cells, has been observed in the joints of RA patients. This isbelieved to lead to an increased differentiation and activity ofosteoclasts (OCs). It was decided, based on these observations, toinitiate a functional genomics effort in order to identify factors thatmodulate the expression of OPG in primary human synovial fibroblastsderived from RA patients (RASF). The following assays, when used incombination with arrayed adenoviral shRNA (small hairpin RNA) expressionlibraries (the production and use of which are described in WO99/64582),are useful for the discovery of factors that modulate the capacity ofsynovial fibroblasts (SFs) to produce OPG. Candidate factors arefiltered first through a primary assay (further referred to as the “OPGassay”) followed by a secondary assay. These factors can be used as thebasis for the development of novel therapies for the protection,maintenance, or stabilization of bone in rheumatoid arthritis,osteoporosis and also to reduce metastasis of cancer cells to bone.

Example 1 describes the development and setup of the primary assayscreen of an adenoviral siRNA library using an ELISA for detection ofprotein levels of osteoprotegerin (OPG), and is referred to herein asthe “OPG assay”.

Example 2 describes the screening and its results.

Example 3 describes the 3M01 rescreen procedure.

Example 4 describes the setup of the secondary assay, referred to as theOC-RASF-coculture assay.

Example 5. describes the validation of the OPG hits in theOC-RASF-coculture assay.

Example 6 describes the determination of the endogenous expressionlevels of the target genes in RASFs.

Example 7 describes the confirmation of the OPG upregulation withindependent Ad5-siRNAs.

Example 8 describes the determination of the anti-inflammatory effectsof TARGETS.

Example 9 describes the OPG dependency of Ad-siRNA-mediated inhibitionof osteoclast differentiation.

Example 1 Design and Setup of a High-Throughput Screening Method for theIdentification of Regulators of OPG Expression by RASFs

The OPG assay that has been developed for the screening of theSilenceSelect® collection has following distinctive features:

-   -   1) The assay is run with primary human synovial fibroblasts, but        with minimal adaptations, could be used for any other source of        primary cells or even cell lines susceptible to express OPG.    -   2) The assay has been optimized for the use with arrayed        adenoviral collections for functional genomics purposes.    -   3) With minimal adaptations, the assay can also be used to        screen compounds or compound collections.    -   4) The assay can be run in high throughput mode.

The protocol of the OPG ELISA is described below. This protocol is theresult of the testing of various antibodies and various protocols:

The supernatant of cultures of primary human synovial fibroblastsderived from RA patients (RASF cultures) to be analysed is diluted 8times in dilution buffer (1×PBS+0.1% BSA), and 35 μL is transferred to apre-coated ELISA plate. The coating of this plate is performed asfollows: a capture antibody (anti-hOsteoprotegerin Purified MouseMonoclonal IgG2A (Clone 69127.1), R&D systems, Cat.No. MAB8051, 500μg/mL) is diluted to 1 μg/mL in PBS. 40 μL, of this dilution is addedper well and an overnight incubation is performed at 4° C. The next day(day 2), the plates are washed once with PBST (1×PBS with 0.5% Tween)and once with 1×PBS (GIBCO). After the washes, the plates are blockedwith 100 μL blocking buffer (1% BSA, 5% sucrose, 0.05% NaN₃) for 4hours. Another wash step is performed with PBST, and a detectionantibody mixture (50 ng/mL) is then added to the plates. This detectionmixture contains following detection antibody: biotinylated hOPGaffinity purified Goat IgG (R&D systems Cat.No. BAF805) diluted inPBS+1% BSA. The plate is then stored in the dark for 2 hours and Afterthis incubation time, 2 wash steps are performed, the first with PBSTand the second with PBS. In every well, 35 μL of a streptavidine-HRPconjugate (BioSource Cat.No. SN2004) is added. This conjugate is diluted1/3000 in 1×PBS supplemented with 1% BSA before addition. After a 45 minincubation step, 2 wash steps are performed, the first with PBST and thesecond with PBS. The PBS is then removed and 50 μL POD chemiluminescencesubstrate (Luminol (POD Roche, 1582950) is added to the plates. After a5 min incubation in the dark, readout is performed on a LumiAscentluminometer (Labsystems), PMT default voltage, 100 msec read time.

An example of the performance of the control plate is shown in FIG. 2.Recombinant OPG (R&D cat185-05-025) is diluted from 25 μg/mL to 8 ng/mLin dilution buffer (PBS+0.1% BSA). After that, a 2-fold dilution ismade, also in the dilution buffer to become the standard curve. Thesamples are then tested in the OPG ELISA according to the protocoldescribed above. A dose-dependent increase in the signal detectedindicates the quality of the assay developed.

Example 2 Screening of 11330 “Ad-siRNA's” in the OPG Assay

The OPG assay, the development of which is described in Example 1, hasbeen screened against an arrayed collection of 11330 differentrecombinant adenoviruses mediating the expression of shRNAs in primaryhuman synovial fibroblasts derived from RA patients (RASFs). TheseshRNAs cause a reduction in expression levels of genes that containhomologous sequences by a mechanism known as RNA interference (RNAi).The 11330Ad-siRNAs contained in the arrayed collection target 5046different transcripts. On average, every transcript is targeted by 2 to3 independent Ad-siRNAs. The principle of the screening is illustratedin FIG. 3. In brief, synovial fibroblasts (SFs) are seeded in 384 wellplates and they are infected the day after seeding with the arrayedshRNA library, whereby each well is infected with one individualAd-siRNA. Five days after infection, the medium on the cells isrefreshed and cells are subject to a further incubation of 2 days. Twodays later, supernatant is collected and subjected to the OPG ELISA.

RASFs, passage 1, were acquired from Cell Applications, Inc. andcultured in DMEM medium (Invitrogen), supplemented with 10% fetal bovineserum (HyClone), 100 units/ml penicillin (Invitrogen) and 100 μg/mLstreptomycin (Invitrogen), and incubated at 37° C. and 10% CO₂ untilpassage 6. The cells are typically passaged once a week by a 1/3 split.At passage 6, a “master cell stock” is generated that is stored inliquid nitrogen. Cells from this master stock are used for the completescreening. When a cell stock is thawed, cells are subcultured and usedfor experiments between passages 10 and 12.

For screening, RASFs are seeded in transparent 384 well plates (Greiner)coated with 0.1% gelatin (Merck) at a density of 1000 cells/well in 50μL Synovial Cell growth medium (Cell Applications,

-   Inc.). One day post seeding, 2.5 μL Ad-siRNA virus from each well of    the SilenceSelect® collection (WO 03/020931), stored in 384 well    plates (estimated titer of 2.5×10⁹ viral particles per mL) is    transferred with the aid of a 96/384 channel dispenser (Tecan    Freedom 200 equipped with TeMO96, TeMO384 and RoMa, Tecan A G,    Switzerland) to individual wells of the 384 well plates containing    SFs. Five days after infection, the medium is removed with a    VacuSafe device (Integra) and 80 μL of aMEM (Invitrogen)+10%    FBS(HI)+Pen/Strep medium is added to the wells by means of a    Multidrop. After two days incubation, the supernatant was collected    in 384 well plates (Greiner) and stored at −80° C. till further    processing in the OPG ELISA (described in Example 1). For analysis,    35 μl of an 8-fold dilution—made by transfer of 8.57 μL supernatant    into 384-wells filled with 60 μL dilution buffer (PBS 1×+1% (w/v)    BSA)—is subjected to the OPG ELISA. Dilution and transfer to the    ELISA plate is performed with the TECAN Freedom workstation.

A 384 well control plate is generated to assess the quality of eachassay. The control plate is run in parallel with and under the sameconditions as the aliquot plates from the SilenceSelect® collectionduring the different screening runs. The composition of this plate isshown in FIG. 4, upper panel. The control plate contains control virusesthat are produced under the same conditions as the SilenceSelect®adenoviral collection. The viruses include three sets of negativecontrol viruses (N₁ (Ad5-eGFP_v1_KI), N₂ (Ad5-Luc_v13_(—)1(D), N₃(Ad5-eGFP_v5_(—)1(D)), arranged in diagonal, interspaced with positivecontrol viruses (P₁═P₃=P₃ (Ad5-OPGv1_(—)1(I), B1: blanco, uninfected).Every well of a control plate contains 50 μl of virus crude lysate.Multiple aliquots of this control plate are produced and stored at −80°C. A representative example of the layout and performance of the controlplate tested with the screening protocol described above is shown inFIG. 4, lower panel. In this figure, the layout of the control plate isindicated (upper panel) and the raw OPG signal detected upon performingthe assay for every recombinant adenovirus on the plate is shown in thelower panel. When the value for the OPG level exceeds the cutoff value(defined as 2.6 fold the standard deviation over the negative controls),the format of the field in the table is black background, whitecharacters.

The complete SilenceSelect® collection (11330 Ad-siRNAs targeting 5046transcripts, contained in 30 384 well plates) is screened in the OPGassay according to the protocol described above in two phases. In thefirst screening round (screen A), 7 virus library plates are screenedand rescreened in single, resulting in 2 datapoints for every Ad-siRNA.In a second screening round (screen B) the remaining 23 virus libraryplates are screened in duplicate on independent assay plates in both aprimary and an independent rescreen. As such, 4 datapoints are thusobtained for every Ad-siRNA in screen B. Ad-siRNA viruses are nominatedas primary hits if half of the data points scored above threshold.Threshold settings for screen A and screen B are set at average of alldata points per plate plus 2.6 times standard deviation over all datapoints per plate. A total of 271 hits (80 out of screen A and 193 out ofscreen B) were isolated that scored above the threshold.

In FIG. 5, all datapoints obtained in the screening of theSilenceSelect® collection in the OPG assay are shown. The averagedrelative luminescence data obtained from the duplicate samples in theprimary screen (PS) is plotted against the averaged relativeluminescence data for the corresponding Ad-siRNA obtained in therescreen (DS). The threshold (2.6 times standard deviation) is indicatedby dotted lines. The data for the most particular targets are shown asfilled circles, the data for Ad-siRNA's nominated as hits are indicatedas filled triangles, the data for the non-hit Ad-siRNA's are indicatedas crosses. The strong symmetry observed between the data of the primaryscreen and that of the rescreen (the datapoints are concentrated arounda straight line) demonstrates the quality and reproducibility of thescreening. The relative OPG expression levels obtained in the primaryscreens for all targets, expressed in terms of “fold standard deviationabove plate average”, are listed in Table 3 below.

TABLE 3 The relative OPG expression levels obtained in the primaryscreens for all targets, expressed in terms of “fold standard deviationabove plate average” Primary screen Double screen Hit # 1 2 1 2 H51-0822.751 2.666 4.204 4.216 H51-054 2.245 n/a 2.679 n/a H51-104 3.385 3.2662.719 2.53 H51-172 3.175 4.384 3.916 5.817 H51-181 6.299 6.04 4.7364.661 H51-225 1.635 1.563 3.322 3.365 H51-236 1.742 1.435 2.755 3.245H51-240 1.507 2.316 3.818 3.12 H51-137 4.012 3.055 2.765 2.33 H51-1215.804 5.205 5.981 5.477 H51-122 3.611 3.201 3.274 3.653 H51-014 2.88 n/a3.453 n/a H51-018 3.598 n/a 1.503 n/a H51-040 2.985 n/a 4.703 n/aH51-046 5.154 n/a 6.711 n/a H51-142 3.058 2.758 1.605 1.711 H51-1033.183 3.134 1.729 2.077 H51-119 2.645 2.616 3.456 2.28 H51-145 3.3184.329 2.654 3.524 H51-153 3.332 3.519 3.57 2.16 H51-177 2.61 2.559 3.263.02 H51-183 4.587 3.653 3.665 2.972 H51-206 2.576 2.753 2.511 3.351H51-251 2.016 2.266 3.387 3.512 H51-270 1.516 1.376 2.76 3.182 H51-2612.067 1.979 2.664 3.086

In this primary screen certain targets are obtained, which have beenidentified previously in rheumatoid arthritis (RA) and/or osteoporosis(OP) independently designed and unrelated screens. This serves tovalidate these polypeptides as RA/OP targets and demonstrates theaccuracy and relevance of the screen utilized herein. These targetsrelate to hits H51-103, H51-119, H51-145, H51-153, H51-177, H51-183,H51-206, H51-251 and H51-270. Their nucleic acid sequences are given inSEQ ID NO: 30-39 and their amino acid sequences are SEQ ID NO: 70-79,and their appropriate particulars are provided in Tables 1-5 herein.These are described in WO 2005/063976, WO 2005/121778 and WO 2005/124342each and all of which are incorporated herein by reference.

Example 3 Three MOI Rescreen of the Primary Hits Using IndependentRepropagation Material

To confirm the results of the identified Ad-siRNA in the OPG ELISA thefollowing approach may be taken: the Ad-siRNA hits are repropagatedusing PerC6 cells (Crucell, Leiden, The Netherlands) at a 96-well platelevel, followed by retesting in the OPG assay at three MOIs(multiplicity of infection). First, tubes containing the crude lysatesof the identified hit Ad-siRNA's samples are picked from theSilenceSelect® collection and rearranged in 96 well plates together withnegative/positive controls. The primary hits from screen A and screen Bare each rearranged over four 96-well plates. As the tubes are labeledwith a barcode (Screenmates™, Matrix technologies), quality checks areperformed on the rearranged plates. To propagate the rearranged hitviruses, 40.000 PerC6.E2A cells are seeded in 200 μL of DMEM containing10% non-heat inactivated FBS into each well of a 96 well plate andincubated overnight at 39° C. in a humidified incubator at 10% CO₂.Subsequently, 2 μL of crude lysate from the hit Ad-siRNA's rearranged inthe 96 well plates as indicated above is added to the PerC6.E2A cellsusing a 96 well dispenser. The plates may then be incubated at 34° C. ina humidified incubator at 10% CO₂ for 7 to 10 days. After this period,the repropagation plates are frozen at −20° C., provided that completeCPE could be seen. The propagated Ad-siRNAs are rescreened in the OPGassay at 3 MOI's (4 μL, 2 μL and 1 ML). Infection at 3 MOIs is carriedout as follows: using the 96/384 TeMo pipettor, a ½ and ¼ dilution ismade of each 96-well plate that contains the crude lysate ofrepropagated hits. Subsequently, an aliquot of each of the four 96-wellplates containing the undiluted crude lysate of the repropagated hits ofscreen A or screen B are transferred to one 384-well plate. Similarly,aliquots of the ½ or ¼ (respectively) dilutions are combined into one384-well plate resulting in three 384 well plates containing undiluted,½ or ¼ diluted crude lysates of repropagated hits of screen A (or ofscreen B). Finally, 4 μL of each of these three 384-well plates istransferred to the assay plates, resulting in the 4 μL, 2 μL and 1 μLinfections. Within one 3 MOI rescreen, infections at each MOI isperformed in duplicate, with each singular on a different assay plate.

For most of the primary hits (screen B) the identified Ad-siRNAs areretested in two independent three MOI rescreens. Data analysis for eachof the three MOI screen is performed as follows. For every plate theaverage and standard deviation is calculated for the negative controlsand may be used to convert each data point into a “cutoff value” thatindicates the difference between the sample and the average of allnegatives in terms of standard deviation of all negatives. For each MOI,a threshold setting is defined as the minimal “cutoff value” at whichnone of the negatives would score positive. Threshold settings for thefirst three MOI rescreen are 2.3-2.0-2.0 (for each MOI 4 μL/2 μL/1 μL,respectively). Threshold settings for the second 3 MOI rescreen are2.2-2.4-2.3 for each MOI respectively. Within one of the three MOIrescreens the Ad-siRNA must to score in duplicate in at least one MOI tobe positive. Hits that are found to be positive in both 3 MOIexperiments are defined as “confirmed OPG hits”. 159 of the 193 primaryhits (screen B) are confirmed in this way.

For a minority of the primary hits (screen A) the identified Ad-siRNAsare retested in only one 3 MOI rescreen. Cutoff settings here are asfollow 5-3.6-3.2 (for each MOI 4 μL/2 μL/1 μL respectively) and arebased on 2 negative controls (N2 and N3). 63 out of the 80 primary hitsfrom screen A are found to score in duplicate in at least one MOI withinthis 3 MOI rescreen and are confirmed.

In summary, 222 out of the 273 primary hits (or 81.3%) are confirmedusing repropagated Ad-siRNA material. The 3M01 screening data aresummarized in Table 4: overview of performance of primary OPG hitsidentified in screen A (A) or screen B (B) in further validationexperiments: OPG 3M01 retesting (this example) and testing in cocultureOC assay (see next example). Each of these validation experiments isdone at 3 MOIs (duplicates within one MOI)(*). The table indicates thenumber of MOIs at which a hit scored in duplicate above the cutoffsetting (for OPG) or below the cutoff setting (for the osteoclast assay,OC) for each repeat that is performed (RUN A, B, C). The table alsoindicates if a hit is confirmed (1) or not (0) within each of thevalidation experiments according to hit calling criteria outlineddiscussed above.

All data for the most particular targets obtained in the OPG three MOIretesting (this example) and testing in coculture OC assay (see nextexample) are shown in FIG. 9B. This figure summarizes the cutoffsettings and performance of some primary OPG hits identified in screen A(A) or screen B (B) in further validation experiments: OPG 3MOIretesting and testing in coculture OC assay. Values having cutoffsetting above cutoff value (for OPG assay) or below cutoff value (for OCassay) are indicated by gray shading.

A quality control of target Ad-siRNAs is performed as follows: TargetAd-siRNAs are propagated using derivatives of PER.C6© cells (Crucell,Leiden, The Netherlands) in 96-well plates, followed by sequencing thesiRNAs encoded by the target Ad-siRNA viruses. PERC6.E2A cells areseeded in 96 well plates at a density of 40,000 cells/well in 180 μlPER.E2A medium. Cells are then incubated overnight at 39° C. in a 10%CO₂ humidified incubator. One day later, cells are infected with 1 μl ofcrude cell lysate from SilenceSelect® stocks containing targetAd-siRNAs. Cells are incubated further at 34° C., 10% CO₂ untilappearance of cytopathic effect (as revealed by the swelling androunding up of the cells, typically 7 days post infection). Thesupernatant is collected, and the virus crude lysate is treated withproteinase K by adding to 4 μL Lysis buffer (1× Expand High Fidelitybuffer with MgCl_(2 (Roche Molecular Biochemicals, Cat. No) 1332465)supplemented with 1 mg/mL proteinase K (Roche Molecular Biochemicals,Cat No 745 723) and 0.45% Tween-20 (Roche Molecular Biochemicals, Cat No1335465) to 12 μL crude lysate in sterile PCR tubes. These tubes areincubated at 55° C. for 2 hours followed by a 15 minutes inactivationstep at 95° C. For the PCR reaction, 1 μL lysate is added to a PCRmaster mix composed of 5 μL 10× Expand High Fidelity buffer with MgCl₂,0.5 μL of dNTP mix (10 mM for each dNTP), 1 μL of “Forward primer” (10mM stock, sequence: 5′ CCG TTT ACG TGG AGA CTC GCC 3′) (SEQ. ID NO:110), 1 μL of “Reverse Primer” (10 mM stock, sequence: 5′ CCC CCA CCTTAT ATA TAT TCT TTC C) (SEQ. ID NO: 111), 0.2 μL of Expand High FidelityDNA polymerase (3.5 U/μL, Roche Molecular Biochemicals) and 41.3 μL ofH₂O. PCR is performed in a PE Biosystems GeneAmp PCR system 9700 asfollows: the PCR mixture (50 μL in total) is incubated at 95° C. for 5minutes; each cycle runs at 95° C. for 15 sec., 55° C. for 30 sec., 68°C. for 4 minutes, and is repeated for 35 cycles. A final incubation at68° C. is performed for 7 minutes, 5 μL of the PCR mixture is mixed with2 μL of 6× gel loading buffer, loaded on a 0.8% agarose gel containing0.5 μg/μL ethidium bromide to resolve the amplification products. Thesize of the amplified fragments is estimated from a standard DNA ladderloaded on the same gel. The expected size is approximately 500 bp. Forsequencing analysis, the siRNA constructs expressed by the targetadenoviruses are amplified by PCR using primers complementary to vectorsequences flanking the SapI site of the plPspAdapt6-U6 plasmid. Thesequence of the PCR fragments is determined and compared with theexpected sequence. All sequences are found to be identical to theexpected sequence.

Example 4 Design and Setup of a Screening Method for the Identificationof Regulators of Osteoclast Differentiation in Coculture Background andPrinciple of the Osteoclast Coculture Assay.

FIG. 6A represents the principle of the osteoclast coculture assay. Inthis assay, RASFs are seeded in a multi-well plate. These cells arecapable of expressing factors that modulate the differentiation ofosteoclast precursor cells either in a negative way (e.g. OPG) or in apositive way (e.g. TNF or RANKL). Osteoclast precursor cells are thenseeded on top of the RASFs and M-CSF as well as RANKL are added to thecoculture. In this setting, the osteoclast precursor cells willdifferentiate unless an inhibiting factor is expressed by the coculturedRASFs. As such, this assay allows one to functionally monitor theexpression of factors modulating osteoclast differentiation by RASFs.The readout applied to quantify the differentiation of the osteoclastsin coculture is a cell-based ELISA that measures the expression of amarker specific for differentiated osteoclasts (vitronectin receptor,also called alphav-beta3 integrin). The principle of the screening ofarrayed adenoviral collections in the osteoclast coculture assay isillustrated in FIG. 6B. In brief, RASFs are seeded in multi-well platesand infected with the Ad-siRNA's in an arrayed fashion on day 1. On day7, the osteoclast precursor cells and M-CSF are added on top of theRASFs. Day 8, sRANKL is added and day 19 (after 10 days incubation), thevitronectin cELISA is performed.

4.1 Selection of a Readout for the Osteoclast-RASF Coculture.

Antibody-based detection methods are amenable to HTS development.Therefore, we aimed at evaluating a cELISA detection method for α_(v)β₃integrin (vitronectin receptor) and calcitonin receptor, two markersthat are frequently used to assay OC differentiation and for whichantibodies are commercially available.

Readouts for these markers are evaluated using the commerciallyavailable Poetics™ Osteoclast Precursor Cell System (Cambrex). This cellsystem contains cryopreserved human OC precursors for which, uponthawing and culturing in the provided optimized differentiation medium,differentiate towards mature functional multinucleated OCs. Thesespecific precursor cells will be further referred to as OCPs. AcELISA-based readout with commercially available antibodies for α_(v)β₃integrin and calcitonin receptor is tested (See FIG. 7A). For theexperiment depicted, primary human OCPs cells are seeded at a density of10,000 cells/well in a 96-well plate format and cultured for 10 days inmedium (proprietary Cambrex medium) containing both rRANKL (66 ng/mL)and M-CSF (33 ng/mL) or in medium containing only M-CSF(undifferentiated control) Immunostaining is performed using primaryantibodies for α_(v)β₃ (Monosan) or calcitonin receptor (Serotec) incombination with the Alkaline Phosphatase-Fast Red staining kit (Dako)to visualise the bound primary antibody. The expression of α_(v)β₃integrin and calcitonin receptor is barely detected on undifferentiatedcontrol cultures but is clearly increased if culture conditions allow OCdifferentiation, validating the approach for the detection ofosteoclasts.

While these results prove the feasibility of a cELISA-based measurementof α_(v)β₃ integrin and calcitonin receptor expression to assay OCdifferentiation in monocultures of OCPs, we aim at developing aco-culture assay in which differentiation takes place on top of RASFs.Therefore, we need to show the absence of background signal when RASFcultures, run under conditions suited to OC differentiation, aresubjected to the cELISA procedure for α_(v)β₃ and calcitonin receptordetection. While no background staining is detected for α_(v)β₃, a clearsignal is detected when the cells are assayed for calcitonin receptorexpression (See FIG. 7B). The experiment depicted is performed asfollows. RASF are seeded at 3000 cells/well in a 96-well plate. After 3days of culture, cell layers are fixed and stained using antibodies forα_(v)β₃ and calcitonin receptor, as described below. A clear staining isobserved with the calcitonin receptor mAb, while no signal higher thanbackground (only 2^(nd) Ab or no Ab) when cells are incubated with theα_(v)β₃ mAb. Only the α_(v)β₃ integrin cELISA readout, therefore, isconsidered for further assay development.

In next experiments, proof of principle is delivered for the inhibitionof RANKL induced OC differentiation by RASFs. RASFs (1100 cells/well)are seeded in 384 well plates and infected with either Ad5-eGFP orAd5-OPG (FIG. 7C, panel A) or left uninfected (FIG. 7C, panel B). 24 hrsafter seeding, OC precursor cells (OCP, Cambrex, 1500 cells/well) andM-CSF (40 ng/mL, R&D systems) are added to all wells, as well as therecombinant OPG (rOPG, 22 ng/mL or 66 ng/mL, R&D systems) and IL4 (10ng/mL, R&D systems) (panel B). After one day rRANKL (0 to 60 ng/mL,Cambrex) is added and incubation is performed for 11 days before theα_(v)β₃ integrin cELISA luminescent readout. Luminescence data areexpressed as percentage of the signal obtained for the negative controlsat 15 ng/mL rRANKL concentration (=100%). Results are shown in FIG. 7C.A clear rRANKL dose-dependent induction of OC differentiation isobtained in this experiment (as seen in the uninfected and Ad5-eGFPinfected samples). The differentiation is inhibited by OPG (eitherrecombinant or expressed by SFs). The rOPG dose added (22 ng/mL) issufficient to inhibit the effect of up to 7.5 ng/mL rRANKL. As expected,a higher dose of rOPG is able to inhibit even higher concentrations ofrRANKL: up to 15 ng/mL of rRANKL is efficiently inhibited by 66 ng/mLrOPG. Also rIL4 addition could potently block the rRANKL-driven OCdifferentiation through the inhibition of the RANKL signalisation inpre-OCs. Taken together, this experiment demonstrates that therRANKL-driven OC differentiation process can be blocked by varioussecreted factors and represents a proof of principle experiment for the‘inhibition of RANKL induced OC differentiation assay’.

The protocol of the vitronectin receptor cELISA used for the detectionof osteoclasts in screening setting is as follows:

The medium on top of an osteoclast monoculture or of a osteoclast-RASFcoculture or is removed and 50 μL ice cold MeOH (Riedel-de-Haen, catN^(o) 32213) is added for fixation of the cells. The MeOH is refreshedwith 80 μL MeOH. After incubation for 20 min at −20° C., the MeOH isremoved and the plates were dried in air for 20 minutes. The plates arethen washed twice with 80 μL PBS 1× (GIBCO) and 75 μL of 0.1% caseinbuffer are immediately added to block the plates. The casein buffer isprepared as follows: 2 g casein in 80 mL Milli Q, adjust to PH 12, stir15 min at RT, adding 200 mL 10×PBS, adjust to 2 L Milli Q and adjust toPH7.4. The plates are blocked for at least 2 hours at RT and the caseinbuffer is then removed. 25 μL of EC buffer is then added to the plates.The EC buffer is prepared as follows: 8 g casein, 4.26 g Na₂HPO₄, 4 galbumine bovine, 1.38 g NaH₂PO₄.H₂O, 1 g CHAPS, 46.6 g NaCl in 150 mLMilliQ, 8 mL EDTA pH8, adjusting to pH12, stirring 15 min at low heat,adding 10 mL NaN₃ 10%, adjusting to 2 L with MilliQ, adjusting to pH7.0.The EC buffer is then removed and 35 μL of the primary antibody(Monosan, Mon2033) is added to the plates. The plates are incubatedovernight at 4° C. and then washed twice, once with PBST (1×PBS with0.05% Tween20) and once with 1×PBS. The secondary antibody (2000-folddilution of goat anti-mouse immunoglobulins from DAKO) is then added tothe plate in buffer C. Buffer C is prepared as follows: 0.82 gNaH₂PO₄.H₂O, 4.82 g Na₂HPO₄, 46.6 g NaCl, 20 g Albumin bovine, adjust to2 L with MilliQ, add 8 mL 0.5M EDTA pH8.0, adjusting to pH7.0 andsterilize. An incubation of maximally 1 hour is performed. After theincubation, the plates are washed twice with PBST (1×PBS with 0.05%Tween20) and once with 1×PBS. The read out is performed with Luminol(POD Roche, 1582950), a chemiluminescence substrate.

The vitronectin receptor cELISA is adapted for the screening of anarrayed adenoviral collection on a coculture as follows. Day 1, the RASFcells (1000 cells/well) are seeded on a 0.1% gelatin coated plate(Greiner, cat. N^(o) 781080) in 50 μL medium (Synovial Growth medium,CellApplication). One day later (day 2) the cells are infected with 4 μLof Ad-siRNA material from library (at 3 dilutions). On day 7, the mediumwas refreshed with 30 μL co-culture medium (aMEM, (GIBCO, cat. N^(o)22571-020) supplemented with 10% FBS and a mixture of penicillin andstreptomycin), containing 60 ng/mL rhMCSF (Cambrex; PT-9010). 1250osteoclast precursor cells (Cambrex; Cat. N^(o) 2T-110, contained in 30μl medium) are then added on top of the RASFs. Day 8, sRANKL (Cambrex,osteoclast culture bullet kit) is added to a concentration of 30 ng/mL.On day 19 (after 10 days incubation at 37° C.; 5% CO₂), the vitronectinreceptor cELISA is performed.

Example 5 Validation of the OPG Hits in the Osteoclast-RASF CocultureAssay

Confirmed OPG hits are further analyzed in the osteoclast RASFco-culture assay that is developed and performed described above(Example 4). The desired effect is the following: knock-down of theAd-siRNA target gene expression in the RASFs monolayer should inhibitosteoclast differentiation driven by RANKL and MCSF. For the majority ofthe confirmed OPG hits (hits originating from screen B) testing in theosteoclast differentiation assay is as follows. Ad-siRNA are tested intwo independent experiments, each carried out at 3 MOIs. The virusmaterial for the Ad-siRNA and positive and negative controls is the sameas that prepared for retesting of the primary hits in 3 MOI OPG. Theresults obtained after read out of the osteoclast differentiation assayare converted into “cutoff values” based on the average and standarddeviation of the negative controls on each plate as described for the 3MOI OPG ELISA, except that results for even and odd rows are firstseparated in order to correct for an observed difference in signalstrength of controls on even/odd rows. For each MOI, a threshold forhitcalling is set. The threshold is the lowest “cutoff” value at whichnone of the negatives score positive (i.e. have a cutoff values lowerthan the threshold). Settings in the two independent three MOI OCtestings are −1.8/−1.8/−1.8 (for the 3 MOIs 4 μL/2 μL/1 μLrespectively). The Ad-siRNA is required to score in duplicate in atleast one of the MOIs to be positive within a three MOI experiment. 53of the 159 confirmed OPG hits (screen B) are positive in both three MOIexperiments and passed this control test. 33 others, were positive inonly one of the two three MOI experiments. These 33 Ad-siRNAs are cherrypicked out of the virus plates together with the controls and tested fora 3^(rd) time in the OC assay at 3 MOIs. 7 of the 33 are found to have apositive score after analysis and passed the OC differentiation controltest. Therefore, 60 out of the 159 OPG confirmed hits originating fromscreen B (i.e 37.7%) are thus found to pass the OC co-culture assay.

For a minority of the confirmed OPG hits (originating out of screen A),Ad-siRNAs are tested in 3 independent OC co-culture experiments. One ofthese is performed at only one MOI (2.5 μL infection out ofSilenceSelect® collection tubes) and 2 are performed at 3 MOIs usingrepropagated virus material obtained after primary screening. Thresholdsettings for hitcalling are based on the results of the appropriatenegative controls as described above. To pass the OC differentiationcriterium, Ad-siRNAs are required to have a positive score in 2 out ofthe 3 experiments. Of the 63 confirmed OPG hits, 23 (i.e. 36.5%) passedthis criterium.

In summary, 83 of the 222 confirmed OPG hits (or 37.4%) are also foundto inhibit OC differentiation in the co-culture assay. The resultsobtained in the secondary assay for the TARGETS are summarized in Table4 (screen A and screen B) and the raw data obtained for the particulartargets are shown in FIG. 9 (screen A and screen B).

TABLE 4 Summary of the data obtained for the MOI rescreen and secondaryassay (osteoclast differentiation assay) for all hits Screen A. Data forthe hits from 7 SilenceSelect ® plates: 3 MOI OPG 3 MOI OC RUN A OPG RUNA RUN C OC score @ confirmed score @ RUN B score @ confirmed HIT REFSYMBOL # MOIs Hit #MOIs Hit #MOIs Hit H51-014 MAP3K3 2 1 2 1 2 1 H51-018P2RY14 1 1 1 1 0 1 H51-040 NEK3 3 1 0 1 1 1 H51-046 KLKB1 2 1 2 1 0 1H51-054 MAP4K4 3 1 3 1 2 1 Screen B. Data for the hits from thescreening 23 SilenceSelect ® plates: 3 MOI OPG 3 MOI OC RUN A RUN B RUNA RUN B RUN C score @ score @ OPG score @ score @ score @ OC # #confirmed # # # confirmed HIT REF SYMBOL MOIs MOIs Hit MOIs MOIs MOIsHit H51-082 NTRK2 3 3 1 3 3 NA 1 H51-104 MMP17 3 3 1 3 1 NA 1 H51-121SLC4A8 3 3 1 3 3 NA 1 H51-122 ENPP2 3 3 1 3 2 NA 1 H51-137 MRAS 3 3 1 31 NA 1 H51-142 FNTA 1 2 1 3 1 NA 1 H51-172 PLA2G12A 3 2 1 3 3 NA 1H51-181 MGLL 3 3 1 1 2 NA 1 H51-225 GPR44 3 2 1 3 3 NA 1 H51-236 MIR16 33 1 1 2 NA 1 H51-240 PTK6 3 2 1 2 3 NA 1 H51-103 USP9Y 3 3 1 3 2 NA 1H51-119 CDC7 3 2 1 1 1 NA 1 H51-145 PPIA 3 3 1 3 3 NA 1 H51-153 TOP2B 33 1 2 3 NA 1 H51-177 PPP2CB 3 3 1 3 1 NA 1 H51-183 COX10 3 3 1 2 2 NA 1H51-206 CCR1 3 1 1 1 1 NA 1 H51-251 B3GALT1 3 3 1 2 2 NA 1 H51-261 CXCR63 3 1 3 3 NA 1 H51-270 SLC9A8 2 3 1 2 1 NA 1 NA = not applicable (athird run of the OC assay was not done because it scored in previoustwo). (*) = OC run B for hits identified out of screen A was done atonly 1 MOI in two independent experiments. To be a hit in this run, theAd-siRNA had to score in one of the two experiments.

Example 6 Analysis of the Expression Levels for Certain TargetsIdentified in Human Primary Synovial Fibroblasts Derived from Synoviumof RA Patients

Expression levels for certain identified targets are determined indifferent isolates of primary human synovial fibroblasts as follows.

The RASFs isolates are obtained as cryo-preserved passage 2 cells fromCell Applications Inc. (Cat. No. 404-05). These cells are cultured andpropagated in DMEM (Invitrogen) supplemented with 10% (v/v)heat-inactivated FBS (ICN) and 1× Pen/Strep (Invitrogen). For expressionanalysis, cells are cultured to passage 11.

For RNA preparation, the primary human synovial fibroblasts are seededin 10-cm Petri dishes (500,000 cells/dish) in 6-well plates. Afterovernight incubation, medium is refreshed with 6 mL of M199 mediumsupplemented with 1% (v/v) heat-inactivated FBS containing 1× Pen/Strep.24 hours later, total RNA is extracted using the “SV Total RNA Isolationkit” (Promega).

The concentration of RNA in each sample is fluorimetrically quantifiedusing the “Ribogreen RNA quantitation kit” (Molecular Probes). A similaramount of RNA from each preparation is reverse transcribed into firststrand cDNA with the “Taqman reverse transcription kit” from AppliedBiosystems. Briefly, 40 ng RNA is included per 20 μL reaction mixcontaining 50 pmol of random hexamers, 10 U Rnase inhibitor, 25 UMultiscribe reverse transcriptase, 5 mM MgCl₂ and 0.5 mM of each dNTP.The reaction mixture is incubated at 25° C. for 10 minutes, followed by30 minutes incubation at 48° C. and heat inactivation (5 minutes 95° C.)of the reverse transcriptase in a thermocycler (Dyad, MJ Research).Reactions are immediately chilled to 4° C. at the end of the program. Toavoid multiple freeze/thaw cycles of the obtained cDNA, the differentsamples are pooled in 96-well plates, aliquoted and stored at −20° C.

Real-time PCR reactions are performed and monitored using the “ABI PRISM7000 Sequence Detection System Instrument” (Applied Biosystems).Pre-designed, gene-specific Taqman probe and primer sets forquantitative gene expression are purchased from Applied Biosystems aspart of the “Assays on Demand” Gene expression products. Thesecommercially available kits are quality checked by the supplier andallow quantitative determination of the amount of target cDNA in thesample. The “Assays on Demand” gene expression products are usedaccording to the protocol delivered by the supplier. The PCR mixtureconsisted of 1×“Taqman Universal PCR Mastermix no AmpErase UNG” and 1×“Taqman Gene Expression Assay on Demand mix” and 5 μL of theretro-transcription reaction product (1-100 ng of RNA converted intocDNA) in a total volume of 25 μL. After an initial denaturation step at95° C. for 10 minutes, the cDNA products are amplified with 40 cyclesconsisting of 95° C. for 15 sec, and 60° C. for 1 minute. To normalizefor variability in the initial quantities of cDNA between differentsamples, amplification reactions with the same cDNA are performed forthe housekeeping gene β-actin using the predeveloped β-actin “Assays ondemand” primer set and Taqman probe mix and “Taqman Universal PCRMastermix” (all Applied Biosystems) according to the manufacturer'sinstructions. To identify any contamination resulting from residualgenomic DNA, real-time PCR reactions with product from a control (—RT)reverse transcription reaction that is performed under the sameconditions but without the addition of the reverse transcriptase areincluded for each sample. Threshold cycle values (Ct), for example, thecycle number at which the amount of amplified gene of interest reached afixed threshold are determined for each sample. For each sample, the ΔCtvalue is determined by subtracting the Ct value of the endogenouscontrol (β-actin) from the Ct value obtained for the target gene. A geneis considered as expressed in primary human SFs if the ΔCt valueobtained for this hit is lower than 13.3 in at least one of theavailable 2 synovial isolates, activated or not. Genes with a ΔCt valuebelow 9.9 are considered highly expressed in RASFs. The results of theexpression profiling experiments are summarized in Table 5. The ΔCtvalue relative to β-actin obtained for various targets in 2 isolates ofuntriggered SFs are given in this Table 5.

TABLE 5 Determination of the Relative Expression Levels of the TARGETSin Primary Synovial Fibroblasts by Real-Time PCR RASF cells - RASFcells - THP1 SEQ Accession Assay on Untriggered Triggered expressedTarget ID ID # No. demand Ct DCt (*) Ct DCt (*) in RASFs KLKB1 25NM_000892 Hs00168478_m1 35.09 13.68 35.15 13.64 yes ENPP2 19 NM_006209Hs00196470_m1 23.78 2.37 23.95 2.44 yes FNTA 26 NM_002027 Hs00357739_m124.69 3.28 24.86 3.35 yes MAP3K3 20 NM_002401 Hs00176747_m1 26.39 4.8126.29 4.58 yes MAP4K4 6 NM_004834 Hs00377415_m1 24.47 2.89 24.53 2.82yes MMP17 10 NM_016155 Hs00211754_m1 28.79 7.21 28.32 6.61 yes GPR44 14NM_004778 Hs00173717_m1 # 38.15 16.64 MGLL 13 NM_007283 Hs00200752_m124.11 2.7 23.55 2.04 yes MRAS 17 NM_012219 Hs00171926_m1 25.96 4.3826.78 5.07 yes PLA2G12A 11 NM_030821 Hs00830106_s1 27.34 5.76 27.57 5.86yes MIR16 15 NM_016641 Hs00213347_m1 26.45 4.87 26.85 5.14 Yes NTRK2 1NM_006180 Hs00178811_m1 25.23 3.82 25.49 3.98 Yes NEK3 23 NM_002498Hs00300928_m1 31.11 9.53 31.66 9.95 Yes PTK6 16 NM_005975 Hs00178742_m135.36 15.94 35.26 15.97 Yes SLC4A8 18 NM_004858 Hs00191516_m1 30.08 8.530.8 9.09 Yes CXCR6 40 NM_006564 Sybr Green 28.45 6.87 29.76 8.05 YesPrimers CCR1 37 NM_001295 Hs00174298_m1 36.96 15.55 38.07 16.56 Yes

Example 7 “On Target Analysis” Using KD Viruses

To strengthen the validation of a hit, it is helpful to recapitulate itseffect using a completely independent siRNA targeting the same targetgene through a different sequence. This analysis is called the “ontarget analysis”. In practice, this is done by designing multiple newshRNA oligonucleotides against the target using a specialised algorithmdescribed, and incorporating these into adenoviruses according to WO03/020931. After virus production, these viruses are arrayed in 96 wellplates, together with positive and negative control viruses. On average,6 new independent Ad-siRNAs are produced for a set of targets. Twoindependent repropagations of these virus plates are then performed asdescribed above for the 3 MOI rescreen. The plates produced in these 2independent repropagations are tested in the OPG assay at 3 MOIS and induplicate in 2 independent experiments according to the protocoldescribed for the 3 MOI rescreen (Example 3). Ad-siRNAs mediating anincrease in OPG levels above the set cutoff value in at least 1 MOI inthe 2 independent experiments are nominated as hits scoring in the “ontarget analysis”. The cutoff value in these experiments is defined asthe average over the negative controls+2 times the standard deviationover the negative controls. Through this exercise, the following mostparticular targets are identified: ENPP2, CXCR6, MAP3K3, PTK6, MRAS. Thedata obtained for these targets in one of the “on target analysis” testsare shown in FIG. 8. In this Figure, the raw data obtained in thedetermination of the OPG levels are shown. For every target, the averageof the raw OPG data obtained for the negative controls tested on thesame plate are shown and allow to appreciate the increase in OPGexpression for the hit Ad-siRNAs.

Example 8 Determination of the Anti-Inflammatory Effects of OPG Targets

In addition to the bone erosion aspect described above, rheumatoidarthritis has also a strong inflammatory component, as indicated by theefficacy of TNFα blocking agents. To further strengthen the profile of aselection of OPG hits an additional investigation may be performed. Theaim of this exercise is to demonstrate, besides the OPG-inducing andthus bone-protective properties, the additional anti-inflammatorycharacter of these OPG hits. Basically, the additional testing performedis aimed at demonstrating which OPG hits are able to reduce cytokineactivation of RASFs as monitored by the expression of a cytokine-inducedmarker, MMP1. This additional testing allows the identification of morepreferred hits. This additional testing may be performed as follows:

8.1 Virus Collection and Handling:

For a selection of OPG hits targeting expression of a certain gene, aset of independent KD viruses are collected, that mediate the reductionof the expression of the same target gene through different sequences onthe target mRNA. These viruses, together with the original OPG hitviruses, are arrayed in 96 well plates (“hit plates”), together withpositive and negative control viruses. The general layout of the plateis depicted in FIG. 10 h. As the outer wells are left empty to avoidedge effects, every control plate can accommodate 60 samples in total:40 hit viruses and 20 control viruses. A KD virus that targets MMP1 isselected as positive control (4 wells per plate), whereas 3 differenttypes of negative control viruses were used that target eitherluciferase gene transcripts (8 wells per plate), M6PR gene transcripts(4 wells per plate) or eGFP gene transcripts (4 wells per plate). Thereconstituted plates are repropagated to ensure homogeneity of thetiters of the viruses tested.

8.2 Cell Handling and Transduction of RASFs

At day 0, RASFs (with passage number below 11) are seeded in 96 wellplates at a density of 3000 cells/well in 50 μL of medium. One day later(day 1), 8, 16 or 24 μL of the virus crude lysate contained in the virusplates is transferred to the plates containing the cells. As every virusload is tested in duplicate, 6×60 datapoints are generated for every“hit plate” tested.

8.3 Cell Triggering and Supernatant Collection

Five days after transduction of the cells, the reduction in theexpression of the target gene mediated by the KD viruses is fullyeffective. Day 6, medium is removed and replaced by M199 medium+1% FBScontaining an eight-fold dilution of a “TNFα based trigger”. Thistrigger is prepared as follows. The production of the “TNFα basedtrigger” is initiated by seeding THP-1 monocytic cells in M199 mediumsupplemented with 1% serum at a density of 1×10E6 cells/mL. One dayafter seeding, recombinant human TNFalpha (Sigma) is added to theculture flasks to a final concentration of 25 ng/mL. 48 hours afteraddition of the cytokine, the supernatant is collected and stored at−80° C. in aliquots until further use. Every new batch of “TNFα basedtrigger” is characterized for its efficacy at inducing MMP1 expressionby RASFs. This trigger contains a variety of inflammatory mediators thatactivate diverse signal transduction pathways in RASFs. Day 8,supernatant on top of the triggered cells is collected and subjected toa MMP1 ELISA.

8.4 MMP1 ELISA

The MMP1 ELISA is performed in 384 well format as described in WO2006/040357. The following protocol is applied: white Lumitrac 600 384well plates (Greiner) are coated with 2 μg/ml anti-MMP1 antibody MAB1346(Chemicon). The antibody is diluted in buffer 40 (1.21 g Tris base(Sigma), 0.58 g NaCl (Calbiochem) and 5 mL 10% NaN₃ (Sigma) in 1 LmilliQ water and adjusted to pH 8.5). After overnight incubation at 4°C., plates are washed with PBS (80 g NaCl, 2 g KCl (Sigma), 11.5 gNa₂HPO₄.7H₂O and 2 g KH₂PO₄ in 10 L milliQ; pH 7.4) and blocked with 100μl/well Casein buffer (2% Casein (VWR International) in PBS). Next day,casein buffer is removed from ELISA plates and replaced by 50 μL/well ECbuffer (4 g casein, 2.13 g Na₂HPO₄ (Sigma), 2 g bovine albumin (Sigma),0.69 g NaH₂PO₄.H₂O (Sigma), 0.5 g CHAPS (Roche), 23.3 g NaCl, 4 ml 0,5 MEDTA pH 8 (Invitrogen), 5 mL 10% NaN₃ in 1 L milliQ and adjusted to pH7.0). 0.25 mM DTT (Sigma) is added to the thawed samples plates. Afterremoval of the EC buffer, 20 μL of sample is transferred to the ELISAplates. After overnight incubation at 4° C., the plates are washed twicewith PBS, once with PBST (PBS with 0,05% Tween-20 (Sigma)), andincubated with 35 μL/well biotinylated anti-MMP1 antibody solution(R&D). This secondary antibody is diluted in buffer C (0.82 gNaH₂PO₄.H₂O, 4.82 g Na₂HPO₄, 46.6 g NaCl, 20 g bovine albumin and 4 mL0.5M EDTA pH 8 in 2 L milliQ and adjusted to pH 7.0) at a concentrationof 5 μg/mL. After 2 hours of incubation at RT, the plates are washed asdescribed above and incubated with 50 μl/well streptavidin-HRP conjugate(Biosource). Streptavidin-HRP conjugate is diluted in buffer C at aconcentration of 0.25 μg/mL. After 45 minutes, the plates are washed asdescribed above and incubated for 5 minutes with 50 μl/well BM ChemELISA Substrate (Roche). Readout is performed on the Luminoscan AscentLuminometer (Labsystems) with an integration time of 200 msec or with anEnvision reader (Perkin Elmer).

8.5 Hit analysis

The ability of the collected viruses to reduce the expression of MMP1 byRASFs activated with a “TNFα-based trigger” may be determined asfollows. For every plate, 3 control wells are left untriggered, allowingto determine if the MMP1 expression is induced as expected. 17 controlwells (containing 13 negative controls and 4 positive controls) aretriggered. The average and standard deviation is calculated for the MMP1signal over the 13 triggered negative control wells. For everydatapoint, the normalized reduction in MMP 1 expression is calculated asfollows:

Normalized reduction of MMP1 signal for KD virus X=[(Average signal for13 negative controls−signal for KD virus X)/(standard deviation of theMMP1 signal over the 13 negative controls)].

Every datapoint for which the normalized reduction of MMP1 expressionexceeding 2 is considered “positive”, i.e. the “TNFα-basedtrigger”—induced MMP1 expression is considered to be reduced in asignificant way in these samples. For these viruses, the differencebetween MMP1 signal for the virus X and of the negative controls exceeds2 times the standard deviation over the negative controls. As such, 6independent normalized MMP1 datapoints are generated for every tested KDvirus. Viruses for which at least 3 out of the 6 datapoints are“positive” are considered a hit in the MMP 1 assay. A summary of thedata obtained for 7 OPG hits is shown in Table 6. For 6 out of the 7targets tested, at least one KD virus is identified that significantlyreduces “TNFα-based trigger” induced MMP1 expression. As such, theinhibition of the activity of these genes is expected to increase theOPG expression by RASFs and to reduce the response of RASFs toinflammatory cytokines. An example of the data obtained in the MMP1assay is given in FIG. 11.

TABLE 6 Outcome of the additional testing of OPG hits in the “MMP1assay” Nr of constructs inhibiting Target Nr of independent “TNFα-basedtrigger”- Name SEQ ID NOs KD viruses tested induced MMP1 expressionENPP2 19 7 3 GPR44 14 3 2 KLKB1 25 10 3 MAP4K4 6, 7, 8, 9 8 3 MMP17 10 32 NTRK2 1, 2, 3, 4, 5 4 0

For 7 selected OPG hits, up to 9 additional KD virus constructs(“independent KD viruses”) targeting the expression of the same gene arecollected. The number of constructs per OPG hit that mediated asignificant reduction of the “TNFα-based trigger”-induced MMP1expression is indicated in the table.

Example 9 OPG Dependency of Ad-siRNA-Mediated Inhibition of OsteoclastDifferentiation In RASF-Osteoclast Cocultures

In example 5, Ad-siRNA OPG hits are selected based their ability toreduce RANKL-induced osteoclast differentiation in cocultures withtransduced RASFs. The aim of the assay described in this example(further referred to as the “OPG dependency assay”) is to demonstratethat the observed inhibition of osteoclast differentiation in theco-culture assay is due to the increased OPG release by RASFs, that weretransduced with selected Ad-siRNAs. The principle of this assay isdepicted in FIG. 6A. In brief, Ad-siRNAs are tested in the osteoclastco-culture assay with or without inclusion of an anti-OPG antibody thatcan neutralize OPG bioactivity. The desired profile for the Ad-siRNAs isthe following: inhibition RANKL-driven osteoclast differentiation whenthe co-culture assay is performed in absence of the anti-OPG antibodyand absence of effects when the assay is performed in presence of theanti-OPG antibody. The anti-OPG antibody (Cat. N^(o) AF805, R&D Systems)selected for the experiments is a goat polyclonal IgG antibody and asshown in FIG. 7C, is able to neutralize soluble OPG and preventOPG-mediated inhibition of sRANKL-driven osteoclast formation in theco-culture assay. The additional testing is performed as follows:

9.1 Virus Collection and Handing.

Confirmed OPG hits that are also found to inhibit OC differentiation inthe co-culture assay (Example 5) may be selected for testing in the “OPGdependency assay”. The virus material used for this experiment is thesame as that prepared for retesting of the primary hits in the 3 MOI OPG(Example 3). Selected Ad-siRNAs are cherry picked out of these virusplates and re-arrayed in 96-well plates (“hit plates”), together withthe respective positive and negative control viruses (i.e. controlviruses that were repropagated simultaneously with the Ad-siRNAs uponpreparing the material for the 3M01 retesting). The general layout of aplate is depicted in FIG. 4. Every plate contains 4 wells for 3different types of negative control viruses (N1=Ad5-eGFP_v1_KI,N2=Ad5-Luc_v13_(—)1(D), N3=Ad5-eGFP_v5_(—)1(D), and one well containinga positive control (P=Ad5-OPG_v1_KI). In the assay, transduction isperformed as follows: 3 μL out of the virus hit plates are transferred 4times to a 384-well assay plate such that all four quadrants areinfected with the same virus.

9.2 Assay description

Day 1, RASFs cells (1000 cells/well) are seeded on a 0.1% gelatin coated384-well plate (Greiner, cat N^(o) 781080) in 50 μL medium. Thefollowing day (Day 2) the cells are infected with 3 μL of Ad-siRNAmaterial. Transfection is done in quadruplo (all 4 quadrants relative toone 96-well, are infected with the same Ad-siRNA). On day 7, the mediumis refreshed with 30 μL coculture medium and osteoclast precursor cells(1600 cells contained in 30 μL co-culture medium with 105 ng/ml rhMCSF)are then added on top of the RASFs, followed by addition of 10 μL, ofco-culture medium that does not (uneven columns only) or does (evencolumns only) contain the neutralizing anti-OPG antibody at aconcentration of 24 μg/mL. In this way, four datapoints are generatedfor every “hit plate” tested, two in presence and two in absence of theanti-OPG antibody. After overnight incubation (Day 8), 10 μL, ofco-culture medium containing 40 ng/mL rhMCSF and 120 ng/mL sRANKL isadded to all wells to induce osteoclast differentiation. Finalconcentrations of reagents at this time are 15 ng/mL sRANKL, 40 ng/mLrhMCSF and 3 μg/mL anti-OPG antibody (if added). On day 20 (after 11days of incubation at 37° C.; 5% CO₂), osteoclast differentiation isread out by quantifying vitronectin receptor expression by cELISA.

9.3 Hit Analysis

For each hit virus, duplicate values in presence or absence of anti-OPGantibody are averaged and a threshold value was set. Hit viruses thatgenerate values that are under the threshold value are considered toinhibit osteoclast differentiation driven by RANKL. The threshold signalfor hit calling is defined such that none of the values generated byindividual negative controls (in presence or absence of anti-OPG Ab)would score positive. The observed osteoclast inhibition for a hit virusis said to be OPG-dependent, when the averaged value in absence of theanti-OPG Ab is below the threshold and the value in presence of theanti-OPG Ab is above the threshold. An example of the data obtained in arepresentative experiment is given in FIG. 12.

REFERENCES

-   Roodman G D (2004) Mechanisms of Bone Metastasis. N Engl J Med    350:1655-   Pettit A R, Ji H, von Stechow D, Goldring S R, Choi Y, Benoist C,    Gravallese E M (2001) TRANCE/RANKL knockout mice are protected from    bone erosion in a serum transfer model of arthritis. Am J Pathol    159: 1689.-   Pettit A R, Walsh N C, Manning C, Goldring S R, Gravallese    E M. (2006) RANKL protein is expressed at the pannus-bone interface    at sites of articular bone erosion in rheumatoid arthritis.    Rheumatology 45:1068-76.-   Bucay N, Sarosi I, Dunstan C R, Morony S, Tarpley J, Capparelli C,    Scully S, Tan H L, Xu W, Lacey D L, Boyle W J, Simonet W S. (1998)    osteoprotegerin-deficient mice develop early onset osteoporosis and    arterial calcification. Genes Dev. 12:1260-8.-   Kim N, Odgren P R, Kim D K, Marks S C Jr, Choi Y. (2000) Diverse    roles of the tumor necrosis factor family member TRANCE in skeletal    physiology revealed by TRANCE deficiency and partial rescue by a    lymphocyte-expressed TRANCE transgene. Proc Natl Acad Sci USA.    97:10905-10.-   Gravallese E M. (2002) Bone destruction in arthritis. Ann Rheum Dis.    61 Suppl 2:ii84-6.-   Onyia J E, Galvin R J, Ma Y L, Halladay D L, Miles R R, Yang X,    Fuson T, Cain R L, Zeng Q Q,-   Chandrasekhar S, Emkey R, Xu Y, Thirunavukkarasu K, Bryant H U,    Martin T J. (2004) Novel and selective small molecule stimulators of    osteoprotegerin expression inhibit bone resorption. J Pharmacol Exp    Ther. 309:369-79-   Valleala H, Laasonen L, Koivula M K, Mandelin J, Friman C, Risteli    J, Konttinen Y T. (2003) Two year randomized controlled trial of    etidronate in rheumatoid arthritis: changes in serum aminoterminal    telopeptides correlate with radiographic progression of disease. J.    Rheumatol. 30: 468-73.-   Redlich K, Gortz B, Bayer S, Zwerina J, Doerr N, Kostenuik P,    Bergmeister H, Kollias G, Steiner G, Smolen J S, Schett G. (2004)    Repair of local bone erosions and reversal of systemic bone loss    upon therapy with anti-tumor necrosis factor in combination with    osteoprotegerin or parathyroid hormone in tumor necrosis    factor-mediated arthritis. Am J Pathol. 164: 543-55.-   Smolen and Steiner (2003); Lee and Weinblatt (2001); Choy and Panayi    (2001); O'Dell (2004) and Firestein (2003)

From the foregoing description, various modifications and changes in thecompositions and methods of this invention will occur to those skilledin the art. All such modifications coming within the scope of theappended claims are intended to be included therein.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

1. A method for identifying a compound that inhibits bone resorption,comprising: (a) contacting a compound with a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:41-69 and 80, and fragments thereof; and (b) measuring acompound-polypeptide property related to bone resorption.
 2. The methodaccording to claim 1, wherein said polypeptide is in an in vitrocell-free preparation.
 3. The method according to claim 1, wherein saidpolypeptide is present in a mammalian cell.
 4. The method of claim 2,wherein said property is a binding affinity of said compound to saidpolypeptide.
 5. The method of claim 4, which additionally comprises thesteps of: c) contacting a population of mammalian cells expressing saidpolypeptide with the compound that exhibits a binding affinity of atleast 10 micromolar; and d) identifying a compound that inhibits boneresorption.
 6. The method of claim 1, wherein said property isupregulation of a biological pathway producing a biochemical markerindicative of the inhibition of bone resorption.
 7. The method of claim6 wherein said indicator is osteoprotegerin.
 8. The method of claim 1,wherein said property is the activity of said polypeptide.
 9. The methodof claim 1, wherein said property is the expression of said polypeptide.10. The method according to claim 8 or 9, which additionally comprisesthe steps of: c) contacting a population of mammalian cells expressingsaid polypeptide with the compound that significantly inhibits theexpression or activity of the polypeptide; and d) identifying thecompound that inhibits bone resorption.
 11. The method according toclaim 1, which additionally comprises the step of comparing the compoundto be tested to a control.
 12. The method according to claim 11, whereinsaid control is where the polypeptide has not been contacted with saidcompound.
 13. The method according to claim 5 or 10, which additionallycomprises the step of comparing the compound to a control, wherein saidcontrol is a population of mammalian cells that does not express saidpolypeptide.
 14. The method according to claim 1, wherein said compoundis selected from the group consisting of compounds of a commerciallyavailable screening library and compounds having binding affinity for apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 41-69 and
 80. 15. The method according to claim1, wherein said compound is a peptide in a phage display library or anantibody fragment library.
 16. An agent effective in inhibiting boneresorption, selected from the group consisting of an antisensepolynucleotide, a ribozyme, and a small interfering RNA (siRNA), whereinsaid agent comprises a nucleic acid sequence complementary to, orengineered from, a naturally-occurring polynucleotide sequence of about17 to about 30 contiguous nucleotides of a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1-29 and
 40. 17. Theagent according to claim 16, wherein a vector in a mammalian cellexpresses said agent.
 18. The agent according to claim 16, which iseffective in inducing osteoprotegerin (OPG) expression in the OPG assay.19. The agent according to claim 17, wherein said vector is anadenoviral, retroviral, adeno-associated viral, lentiviral, a herpessimplex viral or a sendaiviral vector.
 20. The agent according to claim16, wherein said antisense polynucleotide and said siRNA comprise anantisense strand of 17-25 nucleotides complementary to a sense strand,wherein said sense strand is selected from 17-25 continuous nucleotidesof a nucleic acid sequence selected from the group consisting of SEQ IDNO: 1-29 and
 40. 21. The agent according to claim 20, wherein said siRNAfurther comprises said sense strand.
 22. The agent according to claim21, wherein said sense strand is selected from the group consisting ofSEQ ID NO: 81-97 and
 107. 23. The agent according to claim 20, whereinsaid siRNA further comprises a loop region connecting said sense andsaid antisense strand.
 24. The agent according to claim 23, wherein saidloop region comprises a nucleic acid sequence selected from the groupconsisting of UUGCUAUA or GUUUGCUAUAAC (SEQ ID NO: 108).
 25. The agentaccording to claim 16, wherein said agent is an antisensepolynucleotide, ribozyme, or siRNA comprising a nucleic acid sequencecomplementary to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 81-97 and
 107. 26. A bone resorption inhibitingpharmaceutical composition comprising a therapeutically effective amountof an agent according to claim 16 in admixture with a pharmaceuticallyacceptable carrier.
 27. A method for treatment and/or prevention of adisease involving an imbalance in bone metabolism in a subject sufferingfrom or susceptible to the disease comprising administering to thesubject the pharmaceutical composition of claim
 26. 28. The methodaccording to claim 27 wherein the disease is a joint degenerativedisease.
 29. The method according to claim 28, wherein the disease isrheumatoid arthritis.
 30. A method for treatment and/or prevention of adisease involving abnormal bone resorption in a subject comprisingadministering to the subject the agent of claim
 16. 31. The methodaccording to claim 30, wherein the disease is selected from the groupconsisting of joint degenerative and inflammation diseases.
 32. Themethod according to claim 30, wherein the disease is rheumatoidarthritis.
 33. A method for treatment or prevention of a conditioncharacterized by abnormal osteoprotegrin (OPG) expression and/oractivity in a subject comprising administering to the subject the agentof claim
 16. 34. The method of any of claim 27, 30 or 33 wherein thetreatment and/or prevention additionally comprises administering saidpharmaceutical composition or said OPG inducing agent in combinationwith a disease-modifying anti-rheumatic drug (DMARD) or ananti-inflammatory compound.
 35. The method according to claim 34,wherein said DMARD is selected from the group consisting of Infliximab,Etanercept, Adalimumab, Rituximab, CTLA4-Ig methotrexate, leflunomideand sulfasalazine.
 36. The method according to claim 34, wherein saidanti-inflammatory agent is selected from the group consisting ofcorticosteroids or non-steroidal anti-inflammatory agents.
 37. A methodfor diagnosing a pathological condition involving abnormal boneresorption or a susceptibility to the condition in a subject, comprisingdetermining a first amount of polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 41-67 and 80present in a biological sample obtained from said subject, and comparingsaid first amount with the ranges of amounts of the polypeptidedetermined in a population of healthy subjects, wherein an increase ofthe amount of polypeptide in said biological sample compared to therange of amounts determined for healthy subjects is indicative of thepresence of the pathological condition.